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1ResearchAgreement_CHDI_InstAnimalPhysandGenetics_Ellederova_2016_RecID_A-11609RecID: A-11609

RESEARCH AGREEMENT

RESEARCH AGREEMENT (this "A gr e e m e n t "), dated as of December 1, 2016 (the "E f f ect i ve D a te "), by and between CHDI Foundation, Inc., a New Jersey corporation (the "F ou n d a t i on "), and Institute of Animal Physiology and Genetics ASCR, v.v.i. (the "R e s e a rch I n s t i tut i o n "). The Research Institution and the Foundation shall hereinafter be referred to individually as a "P a rt y " and collectively as the "P a rt i es ".

The Research Institution conducts research in the interest of contributing to and promoting the public good and welfare.

The Foundation supports basic, applied and clinical research aimed at finding diagnoses, treatments, cures and preventions of Huntington's disease.

To further the Foundation's objective, the Foundation desires to fund certain research to be conducted at the Research Institution and, in the interest of the public good and welfare, the Research Institution is prepared to conduct that research.

The Parties have entered into this Agreement for the purpose of, among other things, ensuring that the results of that research are made readily available in a timely fashion to accelerate scientific discovery and facilitate the development of products that diagnose, treat, cure and prevent Huntington's disease.

In consideration of the mutual representations, warranties and covenants contained herein and other good and valuable consideration, the receipt and sufficiency of which are hereby acknowledged, the Parties hereby agree as follows:

Researcher; Research Project

1. Re se ar c her . The "R e se ar c her " means the individual identified as such in App e nd ix A.

2. Resear c h P r o j ec t ; C o n duct o f t h e Resear c h Pr o ject; In t e r im R esear c h P r o j ect Review; C o n t i n ua t i o n o f t h e Resea r ch P r o ject; F o unda t i o n Pr o v i d ed Ma t er i a l s .

(a) Resear c h P r o j ec t . The "Resear c h P r o j ec t " means the program of scientific research described in A pp e n d i x B .

(b) C o n d u ct of t h e R e s e a rch Pro j e c t . Each of the Research Institution and the Researcher will use, or cause to be used, reasonable scientific efforts to conduct the Research Project in accordance with A p p e n d i x B . If at any time the Research Institution or the Researcher makes a good faith determination that (A) the Research Project cannot be conducted substantially in accordance with A pp e n d ix B or the Budget (as defined in Sec t i o n 3(a) of this Agreement) or (B) continued conduct of the Research Project in accordance with Appen d ix B is unlikely to yield scientifically valid or useful results, the Research Institution shall promptly give notice (a "C h a n ge of C i r c u ms t a n ces N o t i ce ") to the Foundation.

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(c) I n ter i m R es e a r ch Pro j e c t R e v i ew; C o n t i n u a t i on of t h e R e s e a rch Pro j e c t . The Foundation shall have the right to elect not to provide financial support for any budget period of the Research Project following the initial budget period of the Research Project by giving written notice (a "D i s c o n t i n ua t i o n o f Fun d ing N o t i ce ") to the Research Institution to such effect at any time prior to that date that is 30 days prior to the end of the then-current budget period of the Research Project.

(d) F o u n da t i o n Pr o v i ded Mate r i a l s .

(i) Obl i ga t i o n to P rov i de F o u n da t i o n Pr o v i ded Ma t er i a l s a n d F o u n da t i o n P r ov i ded M at e r i al In f o r m a t i o n . The Foundation shall be responsible for all aspects of providing, or causing to be provided, to the Research Institution sufficient amounts of those materials expressly identified in A p p e n d ix B (each such material, a "F o u n da t i o n Pr o v i ded Ma t e ri a l ") to be provided to the Research Institution by, or on behalf of, the Foundation to enable the Research Institution to conduct the Research Project. The Foundation shall also be responsible for all aspects of providing, or causing to be provided, to the Research Institution all information and data relating to aFoundation Provided Material that is necessary to enable the Research Institution to conduct the Research Project (all such provided information, the "F o u n da t i o n Pr o v i ded M a t er i a l I n f o r m a t i o n "). The Foundation hereby represents and warrants that all Foundation Provided Materials and Foundation Provided Material Information provided to the Research Institution by, or at the direction of, the Foundation will be provided to the Research Institution in compliance with all applicable federal, state, local and international laws, rules, regulations, orders and guidelines.

(ii) Use a n d Ownership of F o u n da t i o n Pr o v i ded M a t er i a l s and F o u n d a t i o n P r ov i ded M at e r i al In f o r m a t i o n . The Research Institution hereby agrees that the Foundation Provided Materials (including any Foundation Provided Materials contained or incorporated in any substances created in the course of the conduct, or resulting from the performance, of the Research Project) and the Foundation Provided Material Information (A) shall be used by the Research Institution for the sole purpose of conducting the Research Project and for no other purpose (including not using the Foundation Provided Material or Foundation Provided Material Information to attempt to determine, or determine, the identity of any of the person from which the Foundation Provided Material and Foundation Provided Material Information were collected) and (B) shall not, without the prior written consent of the Foundation, be transferred to any third party. Except to the extent required to enable the Research Institution to conduct the Research Project, the Research Institution hereby further agrees that it will not, directly or indirectly, reverse

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engineer, deconstruct or in any way analyze or determine the identity, structure or compositionof any Foundation Provided Materials (including any Foundation ProvidedMaterials contained or incorporated in any substances created in the

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course of the conduct, or resulting from the performance, of the Research Project) or the properties thereof (chemical, biochemical, physical, biological or other). The Research Institution hereby further acknowledges and agrees that (1) all Foundation Provided Material Information shall be deemed Confidential Information (as defined in S e c t i o n 14 of this Agreement) of the Foundation and (2) the Research Institution shall not disclose, reveal, report, Publish or give the Foundation Provided Material Information to any third party. The Research Institution hereby acknowledges and further agrees that a) as between the Research Institution and the Foundation, the Foundation owns the Foundation Provided Materials (including any Foundation Provided Materials contained or incorporated in any substances created in the course of the conduct, or resulting from the performance, of the Research Project) and the Foundation Provided Material Information and b) the Research Institution shall have no ownership or other interest in any Foundation Provided Materials (including any Foundation Provided Materials contained or incorporated in any substances created in the course of the conduct, or resulting from the performance, of the Research Project) orany Foundation Provided Material Information. Immediately upon the earlier to occur of (x) the completion of the Research Project and (y) the termination or expiration of this Agreement, the Research Institution shall appropriately discard or destroy all such unused Foundation Provided Materials and Foundation Provided Material Information.

(iii) Int e ll ectu a l Pr o pe r t y R i g h t s in Re s p ect o f t h e F o unda t i o n Pr o v i ded Ma t er i a l s . The Research Institution acknowledges that the Foundation Provided Materials are or may be the subject of a patent application. Except as provided in this Agreement, no express or implied licenses or other rights are provided to the Research Institution under any patents, patent applications, trade secrets or other proprietary rights of the Foundation, including any altered forms of the Foundation Provided Materials made by the Research Institution. In particular, no express or implied licenses or other rights are provided to use the Foundation Provided Materials (including any Foundation Provided Materials contained or incorporated in any substances created in the course of the conduct, or resulting from the performance, of the Research Project), or any related patents of the Foundation for any purpose other than the conduct of the Research Project. The Research Institution is free to file patent application(s) claiming inventions made by the Research Institution through the use of the Foundation Provided Materials but agrees not to file any patent application containing a composition of matter claim for the Foundation Provided Materials per se.

(iv) No W arrant i e s . Any Foundation Provided Materials and Foundation Provided Material Information provided to the

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Research Institution hereunder are understood to be experimental in nature and may have

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hazardous properties. THE FOUNDATION PROVIDED MATERIALS AND FOUNDATION PROVIDED MATERIAL INFORMATION ARE PROVIDED "AS-IS" AND THE FOUNDATION MAKES NO REPRESENTATIONS AND EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED. THERE ARE NO EXPRESS OR IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR THAT THE USE OF THE FOUNDATION PROVIDED MATERIAL OR FOUNDATION PROVIDED MATERIAL INFORMATION WILL NOT INFRINGE ANY PATENT, COPYRIGHT, TRADEMARK, TRADE SECRET OR OTHER PROPRIETARY RIGHT.

Financial Support

3. Fina n c i a l Support f o r the R es e ar c h Pr o j ect; S o urc e s of F u n d i ng .

(a) Obl i ga t i o n o f t h e F o u n da t i o n to Prov i de F i n a n c i a l Support . The Foundation will provide financial support for the Research Project as provided herein. The amount, purpose, timing and conditions of such financial support (the "Budget")shall be as set forth in A pp e n d i x A . Unless otherwise agreed to by the Foundation pursuant to a notice delivered to the Research Institution in accordance with this Agreement, all work shall be performed and funds expended for the purposes and within the time periods set forth in the Budget. Any funds not so expended or committed shall, unless otherwise agreed, be promptly returned to the Foundation.

(b) S o u r ce s o f F u n d i ng . The Research Institution understands that the Foundation may solicit financial support for the Research Project from third parties andagrees to use any financial support so offered to pay for items in the Budget. Suchfinancial support shall be utilized in accordance with, and subject to, the terms and conditions of this Agreement. Notwithstanding the foregoing, prior to accepting any financial support from any such third party, the Research Institution shall have the right to (i) reasonably request information regarding the source of such financial support to determine whether the acceptance by the Research Institution of such financial support from such third party would violate the stated policies of the Research Institution and (ii) refuse to accept such financial support if in the reasonable determination of the Research Institution the acceptance of such financial support from such third party would violate the stated policies ofthe Research Institution. Any such determination by the Research Institution shallnot relieve the Foundation of its obligations to provide financial support to theResearch Institution in accordance with the terms of this Agreement.

4. Cond i t i o n s to the F o u n da t i o n ' s F i n a n c i a l Supp o rt . The Foundation may, but shall not be obligated to, advance any previously committed financial support for the Research

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Project to the Research Institution upon the occurrence and continuation of any of the following events:

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(a) D e at h , I n c a p a c i t y or E m p l o y m e n t o f R e s e a rc h e r . The Researcher dies, suffers an incapacitating accident or illness, leaves the employ of the Research Institution or takes a sabbatical or leave of absence from the Research Institution;

(b) In t er r upt i o n . The Research Project is interrupted for more than 30 consecutive days at any time, or for more than 45 days in any 12 month period;

(c) Ch a n ge in R e sear c h P r o j ec t . The Research Institution gives a Change ofCircumstances Notice to the Foundation;

(d) L a ck o f A ppr o v a l s . The Research Institution notifies the Foundation that due to lack of necessary approvals it is incapable of performing its obligations under this Agreement;

(e) D u t i es of t h e R e s e a rc h e r . The duties of the Researcher set forth in this Agreement are not fulfilled; or

(f) B r e ach of th i s A gr e e m e n t . There is a material breach of this Agreement by theResearch Institution.

Publication; CHDI Research Group and Results Sharing

5. Def i n i t i o ns . For the purposes of this Agreement, the following terms have the meanings set forth below:

(a) "Publ i sh " means (i) to publish in a peer reviewed scientific journal of general circulation or (ii) present at a scientific meeting and "Pu b l i c a t i o n " has a corresponding meaning.

(b) "Res u l t s " means any scientifically valid methods, data, outcomes or other results made in the course of the conduct, or resulting from the performance, of the Research Project.

(c) "Th i rd P a r ty Re s u l ts " means any scientifically valid methods, data, outcomes or other results (i) made in the course of the conduct, or resulting from the performance, of research conducted by members of the CHDI Research Group (as defined in Sec t i o n 7(a) of this Agreement) (other than the Research Institution and the Researcher) and (ii) funded by the Foundation or one of its affiliates.

6. Pu b lica t i o n . The Researcher shall have (a) the sole and exclusive right to Publish Results and (b) the sole and final authority over any and all decisions related to Publication of Results. The Researcher shall use reasonable efforts to Publish, cause to be Published or otherwise publicly disseminate Results as soon as reasonably possible after such Results have been produced. The Researcher hereby agrees to provide appropriate acknowledgement of the Foundation's support of, and contribution to, the Research Project in any Publication of the Results.

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7. C H D I R e s e a rch Grou p ; S h ar i n g of R esu l ts W i th O t h e rs .

(a) C H D I R e s e a r c h G r o up . The Researcher and the Research Institution hereby acknowledge and agree that they are participating in a community of investigators and organizations (the "CHDI R es e ar c h Gr o up ") funded by the Foundation and its affiliates whose objective is to find diagnoses, treatments, cures and preventionsof Huntington's disease.

(b) D e l i ve r y o f R e s u l t s to t h e Fo u n da t i o n; W i t h dr a w a l of R e s u l t s . The Researcher and/or the Research Institution shall inform the Foundation of all Results produced or discovered within a reasonable period of time following the production or discovery of each such Result. If at any time after informing the Foundation of Results pursuant to this Sect i o n 7(b ) , the Researcher or the Research Institution determines that there is a reasonable scientific basis to conclude that such Results are not scientifically valid, the Researcher or the Research Institution may so notify the Foundation and (i) the Foundation shall take reasonable steps to notify third parties to whom such Results have been disclosed that such Results are no longer scientifically valid and (ii) such Results shall not be deemed to be Results.

(c) D i sc l os u re of R e s u l t s W i t h i n t h e C H D I R e s e a rch G r ou p . The Foundation may disclose Results to any member of the CHDI Research Group who has agreed to each of the covenants set forth in S e ct i on 7( d ) of this Agreement with respect to any Results disclosed to such member.

(d) D i s c l o sure o f T h ird Pa rt y Res u l t s t o t h e Resear c h e r o r t h e Resear c h Ins t i t u t i o n .With respect to any Third Party Results disclosed to the Researcher or the Research Institution, each of the Researcher and the Research Institution hereby agree:

(i) to hold all Third Party Results in confidence until such Third Party Results are Published or otherwise made publicly available (except by breach of this Agreement) so that the disclosure of the Third Party Results among members of the CHDI Research Group does not constitute a public disclosure and so that the ability to patent the Third Party Results is preserved; provided, however, neither the Researcher or the Research Institution shall be required to hold any Third Party Results in confidenceif such Third Party Results (A) were previously known by the Researcher or the Research Institution other than by reason of disclosure by the Foundation; (B) were publicly disclosed except by breach of this Agreement either prior to or subsequent to the receipt of such Third Party Results by the Researcher or the Research Institution; (C) are rightfully received by the Researcher or the Research Institution from a third party without an express obligation of confidence to the Foundation or the member of the CHDI Research Group who discovered

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such Third Party Results; (D) are independently developed by the Researcher or the

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Research Institution without use or reliance upon Third Party Results provided by the Foundation; or (E) are disclosed pursuant to any applicable federal, state, local, or international law, or any judicial or government request, requirement or order, provided that the Researcher or the Research Institution, as the case may be, takes reasonable steps to provide the Foundation with sufficient prior notice in order to allow the Foundation to contest such request, requirement or order;

(ii) to discuss the Third Party Results only with those members of the laboratories of the Researcher that are advised (A) of the confidential nature of the Third Party Results and (B) that the Third Party Results must not be shared with anyone outside of the laboratories of the Researcher until the Third Party Results are made publicly available;

(iii) until the Third Party Results are made publicly available, to not Publish or otherwise publicly disclose methods, data or other results which are derived using the Third Party Results without appropriate written permission; and

(iv) to acknowledge other researchers appropriately if the Third Party Results have contributed to a Publication or presentation of Results.

(e) D i sc l os u re n ot to C o ns t i t u te P u b l i c a t i o n . The Parties acknowledge that it is the intention of the Foundation, the Research Institution and the other members of the CHDI Research Group that the sharing of Results and Third Party Results among members of the CHDI Research Group is to be conducted in a manner so thatsuch sharing shall not constitute "d isc los ur e" for patent purposes.

(f) D i sc l os u re of R e s u l t s O u ts i de t h e C H D I R e s e a rch G r ou p . On and after the date (the "D i s c l o s ure D a te ") which is two years following the End Date specified in A pp e n d i x A , the Foundation shall have the right to disclose (other than through Publication) all Results to any individual or organization without any restrictions unless prior to the Disclosure Date the Researcher or the Research Institution notifies the Foundation that there exists good reasons for such disclosure to be withheld for an additional six-month period, in which case the Disclosure Date will be extended for an additional six months and the provisions of this S e ct i on 7( f ) shall apply to such new Disclosure Date.

Intellectual Property

8. Def i n i t i o ns . For the purposes of this Agreement, the following terms have the meanings set forth below:

(a) "HD R e s e a rch a n d D e v e l o p m e n t " means any activity useful for the creation, development, manufacture or distribution of a product or service for the diagnosis, treatment, cure or prevention of Huntington's disease other than (i)

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the manufacture or distribution of any such product or service for sale or (ii) the sale

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of any such product or service. For the avoidance of doubt, HD Research and Development shall not include any right to (A) manufacture or distribute any such product or service for sale or (B) sell any such product or service (including any transfer of any such services or products made using intellectual property rights, whether or not for consideration, other than a transfer of any such services or products solely for research and development purposes without fee or profit).

(b) "M a d e " when used in relation to any Patentable Invention means the conception or first actual reduction to practice of such Patentable Invention.

(c) "Ot h er HD I n te l l e c tual Prop er t y " means any Other Intellectual Property relating to Huntington's disease or useful for the diagnosis, monitoring, treatment, cure or prevention of Huntington's disease.

(d) "Other I n t e l l e c t u a l Pr o p e rty " means any discovery, invention, formulation, know- how, method, technological development, enhancement, modification, improvement, computer software (including, but not limited to, source code and executable code) and documentation thereof, data or collection of data conceived, discovered or invented in the course of the conduct, or resulting from the performance, of the Research Project which is not a Patentable Invention.

(e) "P a t e n t a b le I n v e nt i o n " means any discovery, invention, formulation, know-how, method, technological development, enhancement, modification, improvement, computer software (including, but not limited to, source code and executable code) and documentation thereof, data or collection of data Made in the course of the conduct, or resulting from the performance, of the Research Project which (i) is or may be patentable or otherwise protectable under Title 35 U.S.C. andcorresponding legislation in other jurisdictions and (ii) is the subject of a patent orpending patent application, including any continuation, continuation-in-part, division, extension, substitute, re-examination, reissue and any other derivative application or patent.

(f) "Patentable HD I n v e n t i o n " means any Patentable Invention relating to Huntington's disease or useful for the diagnosis, monitoring, treatment, cure or prevention of Huntington's disease.

9. O wne rsh ip .

(a) O w n e r sh i p R i g h ts of t h e R e s e a rch I n s t i tut i o n . The Research Institution shall own all intellectual property (including Patentable Inventions and Other Intellectual Property) conceived, discovered, invented or first reduced to practice in thecourse of the conduct, or resulting from the performance, of the Research Project. The Research Institution hereby agrees that it will not sell or otherwise transfer title to any Patentable HD Inventions or Other HD Intellectual Property to any third party unless such third party takes title to such Patentable HD Inventions or Other HD Intellectual Property (i) subject to the rights of the

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Foundation in such Patentable HD Inventions or Other HD Intellectual Property under this Agreement

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and (ii) assumes the obligations of the Research Institution with respect to such Patentable HD Inventions or Other HD Intellectual Property under this Agreement.

(b) O w n e r sh i p R i g h ts of t h e F ou n d a t i o n . Except as expressly set forth in this Agreement, the Foundation shall have no interest in any intellectual property conceived, discovered, invented or first reduced to practice in the course of the conduct, or resulting from the performance, of the Research Project.

10. Inve n t orsh i p . The identity of the inventor of all Patentable Inventions shall be determined in accordance with United States Patent law (or, if the jurisdiction in which patent orother protection is being sought does not permit the application of United States Patentlaw to identify the inventor, then in accordance with the applicable law in that jurisdiction).

11. D i s c l o sure o f Inve n t i o ns; D i s c l o sure o f Pa t e n t Fi li n gs .

(a) Inve n t i o n s . If the Research Institution or the Foundation believes any intellectual property (including Patentable Inventions and Other Intellectual Property) has been conceived, discovered, invented or first reduced to practice in the course of the conduct, or resulting from the performance, of the Research Project, such Party will promptly give notice of such intellectual property to the other Party.

(b) Pa t e n t A p p li c a t i o ns . Within 30 days following the filing of a patent application (including provisional patent applications and each patent application filed corresponding to a previously filed provisional patent application) claiming any Patentable HD Invention, the Research Institution shall give notice to the Foundation setting forth the date of filing of such patent application and shall include with such notice a complete and accurate copy of the patent application filed.

12. No n-Exc lu s ive L icen ses.

(a) No n -E x c l us i ve L i ce ns es of P a te n tab l e H D I n v e n t i o ns . With respect to each patent (including (i) any patent application, divisional, continuation, continuation-in- part, substitute, renewal, reexamination, extension or reissue in respect of such patent or (ii) any intellectual property rights claimed in respect of such patent) claiming a Patentable HD Invention, the Research Institution shall, upon the request of the Foundation, grant to the Foundation a non-exclusive, paid-up, irrevocable, perpetual license throughout the world for HD Research and Development including a license to (A) make, have made, use and have used products or processes resulting from such Patentable HD Invention, (B) practice and have practiced such Patentable HD Invention and (C) use and have used the Confidential Information (as defined in Sec t i o n 14 of this Agreement) relating to such Patentable HD Invention. The foregoing license (1) shall be for HD

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Research and Development only, (2) shall not include any right to manufacture for sale or sell (including any transfer of services or products made using

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intellectual property rights, whether or not for consideration, other than a transfer of services or products solely for research and development purposes without fee or profit), (3) shall not be subject to royalties or other fees and (4) shall includethe right to grant sublicenses on the same terms; provided, that, such sublicense a)is granted without payment of royalties, other fees or profit and b) prohibits the sublicensee from granting sublicenses.

(b) No n -E x c l us i ve L i ce ns es of Ot h er HD I n t e ll e c tual P r ope r t y . With respect to any Other HD Intellectual Property, the Research Institution shall, upon the request of the Foundation, grant to the Foundation a non-exclusive, paid-up, irrevocable, perpetual license throughout the world for HD Research and Development including a license to (i) make, have made, use and have used products or processes resulting from such Other HD Intellectual Property, (ii) practice and have practiced such Other HD Intellectual Property and (iii) use and have used the Confidential Information relating to such Other HD Intellectual Property. The foregoing license (A) shall be for HD Research and Development only, (B) shall not include any right to manufacture for sale or sell (including any transfer of services or products made using intellectual property rights, whether or not for consideration, other than a transfer of services or products solely for research and development purposes without fee or profit), (C) shall not be subject to royaltiesor other fees and (D) shall include the right to grant sublicenses on the sameterms; provided, that, such sublicense (1) is granted without payment of royalties, other fees or profit and (2) prohibits the sublicensee from granting sublicenses.

13. N o n - A s s er t C o v e n a n t . The Research Institution hereby undertakes not to bring any action or assist others in bringing any action, and undertakes to ensure, by contract or otherwise, that its licensees and assignees of any Patentable Invention or Other Intellectual Property will not bring any action or assist others in bringing any action, against the Foundation,its licensees or assignees of any Patentable HD Invention or Other HD Intellectual Property or any other person on the ground that the practice or use, as the case may be, of (a) the inventions described or claimed in any Patentable HD Invention or (b) Other HD Intellectual Property for HD Research and Development infringes or misappropriates the proprietary rights of the Research Institution, its licensees or assignees in any Patentable Invention or Other Intellectual Property.

Confidential Information; Publicity

14. Conf i d e n t i a l I n f o r m a t i o n . For the purposes of this Agreement, the term "C o nf i d e n t i a l In f o r m a t i o n " shall mean this Agreement and all information provided by one Party (the "D is c l os i ng P a rt y ") to another Party (the "R e c e i v i ng P ar t y ") that is clearly identified as "Confidential" by the Disclosing Party at the time of disclosure. If such transmittal occurs orally, the Disclosing Party will promptly reduce such transmittal to writing,

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mark and identify it as confidential, and provide such record to the Receiving Party. Specifically excepted from Confidential Information is all information that: (a) was previously known by the Receiving Party other than by reason of disclosure by the Disclosing Party; (b) is publicly disclosed except by breach of this Agreement either prior to or subsequent to the

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Receiving Party's receipt of such information; (c) is rightfully received by the Receiving Party from a third party without an express obligation of confidence to the Disclosing Party; (d) is independently developed by the Receiving Party without use or relianceupon Confidential Information provided by the Disclosing Party; (e) is disclosed pursuantto any applicable federal, state, local, or international law, or any judicial or government request, requirement or order, provided that the Receiving Party takes reasonable steps to provide the Disclosing Party with sufficient prior notice in order to allow the Disclosing Party to contest such request, requirement or order; or (f) was provided by the Disclosing Party more than five years prior to disclosure by the Receiving Party (or if the confidential nature of the information was specifically reaffirmed in writing by the Disclosing Party during such five-year period, five years after such reaffirmation).

15. Conf i d e n t i a l i t y . The Receiving Party shall not disclose any Confidential Information without prior written authorization from the Disclosing Party, except (a) the Foundation may disclose Confidential Information to the extent expressly permitted by the terms and conditions of S e c t i on 7 of this Agreement; (b) the Foundation may disclose Confidential Information in furtherance of the any license contemplated in Sec t i o n 12 of this Agreement, provided that the Foundation imposes a corresponding obligation of confidentiality on the third party receiving such Confidential Information; and (c) either Party may disclose Confidential Information to the extent expressly permitted by the terms and conditions of S e ct i on 1 6 of this Agreement.

16. P u b l i c i t y . No Party shall use the name, trademarks, logos, physical likeness or other symbol of another Party (or their employees) for any marketing, advertising, public relations or other purposes without the prior written consent of an authorized representative of the affected Party, except that (a) either Party may make reference tothe Foundation's support of the Research Project, provided that, in any such reference, the relationship of the Parties shall be accurately and appropriately described and (b) either Party may disclose, without the other Party's approval, (i) the existence of thisAgreement; (ii) a general summary of the subject matter of the Research Project; (iii) theaggregate dollar amount of financial support to be provided under this Agreement; and (iv) any specific terms of this Agreement that are a matter of public record except by breach of this Agreement.

Representations and Covenants

17. R e p r es e n tat i o n s a n d C o v e n a n ts . The Research Institution hereby agrees to each of the following:

(a) C o m pl i a n ce w i th L a w . The Research Project will be conducted in compliance with all applicable federal, state, local, international, health authority and institutional laws, rules, regulations, orders and guidelines.

(b) R e p ort s . The Research Institution shall provide the Foundation with (i) written progress reports in respect of the Research Project within 30 days of the end of

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each consecutive six-month period during the period beginning on the Start Date specified in A p p e n d ix A and continuing until the End Date specified in A p p e n d ix

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A and (ii) a final written progress report in respect of the Research Project within30 days of the End Date specified in A p p e n d i x A (or, if in accordance withSec t i o n 2(c) of this Agreement funding for any subsequent budget period of the Research Project is not to be provided by the Foundation, the within 30 days of the end of the budget period of the Research Project then being funded by the Foundation). Each progress report shall (A) provide a reasonably detailed description of the status and progress (including a reasonably detailed analysis of milestones achieved or not achieved) of the Research Project for the six-month period covered by such progress report and (B) include a copy of all Results and underlying data.

(c) A u d i t ; Access . The Research Institution shall provide the Foundation with a financial report detailing the use of all funds expended under this Agreement within 60 days of the end of each budget period specified in A pp e n d i x A . At reasonably convenient times and dates, (i) the Foundation and its representatives shall have the right to audit the Research Institution's compliance with this Agreement; provided, however, the Foundation shall not be entitled to exercise such audit rights more than one time during any calendar year and (ii) the Research Institution will provide the Foundation and its representatives with reasonable access to the Research Project facilities, data and personnel (including the Researcher) in order to assess the progress of the Research Project; provided, however, the Foundation shall not be entitled to exercise its access rights more than two times during any calendar year. The Foundation shall be responsible for any expenses incurred by the Foundation in connection with its exercise of the audit and access rights set forth in this S e ct i on 1 7 ( c ) .

(d) P e r m i ts a n d A p p ro v a l s . To the best knowledge of each of the Researcher and the Research Institution, the Research Institution has obtained all, and will use its best efforts to obtain all future assignments, permits, consents and other approvals necessary for the Research Institution to perform its obligations and convey the rights granted under this Agreement.

(e) C on f l i c t i n g O b l i g a t i on s . To the best knowledge of each of the Researcher and the Research Institution, the Research Institution has not granted and will not knowingly grant any right, and has not entered into and will not knowingly enter into any agreement or understanding that conflicts with the Research Institution's obligations or the Foundation's rights under this Agreement.

(f) Cap i t a l Eq u i p m e n t . If (i) the Budget includes funds to purchase property specifically identified as "capital equipment" on A p p e n d i x A and (ii) either (A) the Research Institution ceases to conduct research supported by the Foundation or (B) the Research Institution continues to conduct research supported by theFoundation but no longer requires the use of such capital equipment in connection with such research, then, at the request of the Foundation, the Research Institution shall transfer title to such capital equipment for nominal

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consideration to the Foundation or as the Foundation may otherwise direct. Any such capital

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equipment shall be relocated as directed by the Foundation at the Foundation's expense and risk of loss.

(g) Resear c h M a t e r i a l s . Subject to any other material transfer agreements, theResearch Institution shall, upon the request of the Foundation, make any reagents,cell lines, compounds, animal models or other materials produced in the course ofthe conduct, or resulting from the performance, of the Research Project("Research Ma t e r i a l s ") available to third parties under the terms of the materialtransfer agreement attached hereto as Exhib i t 1 . Subject to any other materialtransfer agreements, the Research Institution shall, upon the request of theFoundation, make any Research Materials available to the Foundation under theterms of the material transfer agreement attached hereto as E x h i b i t 2 .

(h) Res e ar c h Te a m . The Research Project shall only be conducted by individuals(including the Researcher) who have agreed to assign any rights they may acquirein any resulting intellectual property to the Research Institution so that theResearch Institution may perform its obligations under this Agreement. TheResearch Institution shall cause any such individual to assign any such intellectualproperty to the Research Institution so that the Research Institution may performits obligations under this Agreement.

(i) R e s p o n s i b il i t y f o r B r ea c h e s by t he R e s e a r c h e r . The Research Institution hereby acknowledges and agrees that (i) the failure by the Researcher to (A) dischargethe obligations of the Researcher set forth in this Agreement or (B) comply withthe provisions set forth in this Agreement applicable to the Researcher (includingSec t i o n 1 4 and S ec t i o n 1 5 of this Agreement to the extent that the Researcher is aReceiving Party of Confidential Information) shall constitute a breach of thisAgreement by the Research Institution and (ii) the Research Institution shall beliable to the Foundation for any such breach.

(j) Fur t h er Assur a n ces . The Research Institution shall execute such further documents, instruments, licenses and assurances and take such further actions asthe Foundation may reasonably request from time to time to better enable theFoundation to exercise its rights under this Agreement.

Payments; Use of Funds

18. Pa y m e n ts . Subject to the terms and conditions of this Agreement, payments will be remitted to the Research Institution in currency specified in the Budget at the address for payment, or by wire transfer at the account information provided by the Research Institution, as set forth in A p p e n d i x A and shall include a reference to the Researcher.

19. Use of F u n d s . Unless otherwise agreed to by the Foundation pursuant to a notice delivered to the Research Institution in accordance with this Agreement, all funds provided by the Foundation will be used in accordance with the Budget and for no other purpose. Except to the extent expressly set forth in the Budget, the Research

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Institution confirms that it has agreed to waive its indirect and/or overhead costs on the Research

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Project. The Research Institution hereby further agrees that, except to the extent expressly set forth in the Budget, no financial support provided by the Foundation under this Agreement shall be used to pay for the Research Institution's indirect and/or overhead costs.

Term; Termination; Effect of Termination

20. Te r m; Te r m i na t i o n; Effect o f Te r m i na t i o n .

(a) T e r m . The term of this Agreement shall commence on the Effective Date and shall continue in effect until the later to occur of (i) the End Date specified in A pp e n d i x A (or, if in accordance with Sec t i o n 2(c) of this Agreement funding for any subsequent budget period of the Research Project is not to be provided by the Foundation, the end of the budget period of the Research Project then being funded by the Foundation) and (ii) the date on which the final written progress report on the status and progress of the Research Project submitted by the Research Institution pursuant to Se c t i o n 17(b) of this Agreement is approved by the Foundation (such approval not to be unreasonably withheld), unless earlier terminated in accordance with the terms hereof.

(b) T e r mi n a t i o n by t he F o u n d a t i o n . The Foundation may, by giving notice to the Research Institution, elect to terminate this Agreement and discontinue the Research Project upon the occurrence and continuation of any of the following events:

(i) No n - C ur a b l e C o n d i t i on s . The occurrence and continuation of any of the events described in Sec t i o n 4(a) through Se c t i o n 4(d) of this Agreement.

(ii) Brea c h of t h i s A g r e e men t . If the Research Institution or the Researcher defaults in the performance of any of their respective obligations underthis Agreement (including failing to deliver progress reports required to be provided by the Research Institution pursuant to, and in accordance with,Sec t i o n 17(b) of this Agreement) and such default is not remedied within45 days of the receipt by the Research Institution of notice of such defaultfrom the Foundation.

(c) T e r m i n a t i on by t h e R es e a r ch I n s t i tut i o n . The Research Institution may, by giving notice to the Foundation, elect to terminate this Agreement and discontinue the Research Project upon the occurrence and continuation of any of the following events:

(i) Resear c h er . The Researcher dies, suffers an incapacitating accident or illness, or leaves the employ of the Research Institution.

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(ii) B r e ach of th i s A gr e e m e n t . If the Foundation defaults in the performance of any of its obligations under this Agreement and such default is not

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remedied within 45 days of the receipt by the Foundation of notice of such default from the Research Institution.

(d) E ff e c t of Te r mi n a t i o n .

(i) In the event (A) the Foundation elects to terminate this Agreement and discontinue the Research Project pursuant to S e c t i on 2 0 ( b )( i ) of this Agreement or (B) the Research Institution elects to terminate this Agreement and discontinue the Research Project pursuant to Se c t i o n 20(c) of this Agreement, (1) the Foundation shall only be obligated to provide financial support to the Research Institution for the Research Project under this Agreement in an amount necessary to pay for all outstanding non- terminable or non-cancelable obligations (whether such obligation is due and payable before, on or after the date of termination of this Agreement) incurred by the Research Institution up to the date of the occurrence of the event giving rise to the Foundation's right to terminate this Agreement under S ec t i o n 20(b) ( i ) of this Agreement or the Research Institution's right to terminate this Agreement under S e ct i on 2 0( c ) of this Agreement, as the case may be, and (2) the Research Institution shall pay to the Foundation, within 60 days of the date of termination of this Agreement, an amount equal to the amount all funds previously advanced to the Research Institution under this Agreement but unused through the date oftermination of this Agreement in excess of the amount of financial support the Foundation is obligated to provide to the Research Institution under (1) above.

(ii) In the event the Foundation elects to terminate this Agreement and discontinue the Research Project pursuant to S e c t i on 2 0 ( b )( i i ) of this Agreement, (A) the Foundation shall not be obligated to advance any further financial support to the Research Institution for the Research Project under this Agreement; (B) the Foundation shall not be obligated to pay for any outstanding non-terminable or non-cancelable obligations incurred by the Research Institution through the date of termination of this Agreement; and (C) the Research Institution shall pay to the Foundation, within 60 days of the date of termination of this Agreement, an amount equal to the amount all funds previously advanced to the Research Institution under this Agreement but unused up to the date of termination of this Agreement.

(e) F ac i l i ta t i on of C o n t i n ued R e s e a rc h . Upon any termination of this Agreement, if requested by the Foundation, the Research Institution will use its reasonable efforts to facilitate the continuance of the Research Project elsewhere.

(f) Survival o f Cert ain Pro visio ns.

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(i) Lim i t ed Survival of Cer ta i n Pr o v i s i o ns . This Sec t i o n 20( f ) ( i ) (Limited

Survival) and Sec t i o n 14 (Confidential Information), Sec t i o n 15

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(Confidentiality), Sec t i o n 17(b) (Reports), Se c t i o n 17(c) (Audit; Access) and Sec t i o n 20(e) (Facilitation) of this Agreement shall survive the termination of this Agreement for a period of five years.

(ii) Indef i n i t e Survival of Cer ta i n Pr o v i s i o ns . This Se c t i o n 20 ( f ) ( i i ) (Survival) and Sec t i o n 2(d) (Foundation Provided Materials), Sec t i o n 5 (Definitions), S e ct i on 6 (Publication), S e ct i on 7 (CHDI Research Group), S e c t i on 8 (Definitions), S e c t i on 9 (Ownership), S e c t i on 1 0 (Inventorship), S e c t i on 1 1 (Disclosure of Inventions and Patent Filings), S e ct i on 1 2 (Licenses),Sec t i o n 13 (Non-Assert), Section 16 (Publicity), Sec t i o n 17 ( f ) (Capital Equipment), Se c t i on 1 7 (g ) (Research Materials), S e ct i on 1 7 ( i ) (Responsibility for Breaches by the Researcher), S e c t i on 1 7 ( j ) (Further Assurances), Se c t i o n 23 (Notices), Se c t i o n 24 (Indemnity), Sec t i o n 25 (Alternate Dispute Resolution), Se c t i o n 26 (Assignment), Sect i o n 27 (Entire Agreement), Se c t i on 2 8 (No Waiver), S e c t i on 2 9 (Severability), Sec t i o n 30 (Interpretation), Se c t i o n 31 (Governing Law) and Sec t i o n 32 (Strict Construction) of this Agreement shall survive the termination of this Agreement indefinitely.

Miscellaneous

21. I n d e p e n d e nt Co n t r ac to r . The Research Institution is acting as an independent contractor and not an agent, joint venturer or partner of the Foundation.

22. Ind e p e n d e n t Re s earch . Nothing in this Agreement shall be construed to limit the freedom of the Research Institution to engage in similar inquiries made independently under other grants, contracts or agreements with parties other than the Foundation.

23. No t i ces . Any notice required or permitted to be given by this Agreement shall be in writing and shall be delivered by personal delivery, facsimile (provided the sender has evidence of successful transmission) or next day courier service to the Party at its address set forth in A p p e n d i x A or such other address as the Party may, by notice, specify. Any notice so delivered shall be deemed to be given, delivered and received, if delivered by personal delivery, on the day of delivery and if delivered by facsimile or courier service, on the day following dispatch.

24. I nde mn it y.

(a) Inde m n i fica t i o n by t h e Founda t i o n . The Foundation shall indemnify the Research Institution, including, as applicable, its members, trustees, directors, employees and agents, against any and all losses, costs and damages (including reasonable legal fees) suffered by the Research Institution (and/or such other related persons) as a result of the Foundation's negligence, willful misconduct or breach of this Agreement.

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(b) I n d e m n i f i c a t i o n by t he R e s e a r c h I n st i t u t i o n . The Research Institution shall indemnify the Foundation, including, as applicable, its members, directors,

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employees and agents, against any and all losses, costs and damages (including reasonable legal fees) suffered by the Foundation (and/or such other related persons) as a result of the Research Institution's or the Researcher's negligence, willful misconduct or breach of this Agreement.

25. A l t e r n a t i v e D i s pu t e Res o l u t i o n .

(a) M ed i at i o n . If a dispute arises out of or relates to this Agreement, or breach thereof, and the dispute is not resolved by negotiation, the Parties hereby agree to try in good faith to settle the dispute through mediation. Either Party to the dispute may give notice to the other Party of such Party's desire to commence mediation, and a mediation session must take place within 30 days after the date that such notice is given. The Parties must jointly appoint a mutually acceptable mediator. The mediation shall take place in Boston, MA, Chicago, IL, Los Angeles, CA or New York, NY (as is most convenient to both the Parties). If the Parties are unable to agree upon the appointment of a mediator within 10 days after a Party has given notice of a desire to mediate the dispute, either Party may apply in writing to the organization or person agreed to by the Parties in writing,for appointment of a mediator. The Parties further agree to share equally the costs of the mediation, which costs will not include costs incurred by a Party for representation by counsel. If the dispute is not resolved in this manner within 30 days after the commencement of mediation, either Party may submit the disputeto arbitration pursuant to the terms of this Agreement. The Parties agree that any and all such proceedings shall be confidential.

(b) A r b i t r a t i o n . In the event that the parties do not resolve the dispute through mediation as provided above, such dispute arising out of or relating to this Agreement, or breach thereof, shall be settled by a single arbitrator in an arbitration in Boston, MA, Chicago, IL, Los Angeles, CA or New York, NY (as is most convenient to both the Parties) administered by JAMS under (i) its Comprehensive Arbitration Rules and Procedures if the arbitration is held in New York, NY or (ii) the JAMS International Arbitration Rules if the arbitration isheld in Prague, Czech Republic. The award rendered by the arbitrator shall befinal and binding on the Parties, and judgment on the award may be entered in any court having jurisdiction thereof if reasonably necessary for enforcement. The Parties agree that, notwithstanding anything to the contrary in such rules, any and all such proceedings shall be confidential.

26. A s s i g n m e n t . The Research Institution may not assign this Agreement without the written consent of the Foundation. The Foundation may assign this Agreement so long as the assignee expressly assumes in writing the Foundation's obligations in this Agreement.

27. In c o rporat i o n o f A dd e n da, A pp e n d i c es a n d Exhibi t s; Ent i re A gr e e m e n t ; A m e n d m e n t .The addenda, appendices and exhibits identified in this Agreement are incorporated herein by reference and made a part hereof. If anything in any addendum, appendix or

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exhibit attached to this Agreement conflicts with any terms or conditions set forth in the

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body of this Agreement, the terms and conditions set forth in the body of this Agreement shall control. This Agreement constitutes the entire agreement among the Parties relating to the Research Project and all prior understandings and agreements relating to the Research Project are superseded hereby. This Agreement may not be amended except by a document signed by the Research Institution and the Foundation.

28. No W a i v e r . Any failure of a Party to enforce any provision of this Agreement shall not be deemed a waiver of its right to enforce such provision on any subsequent occasion. No waiver of any provision of this Agreement shall be valid unless it is in writing and is executed by the Party against whom such waiver is sought to be enforced. A waiver by any of the Parties of any provision of this Agreement will not be construed to be a waiver of any succeeding breach thereof or of any other provision of this Agreement.

29. S e v e r abi l i t y . Whenever possible, each provision of this Agreement shall be interpreted in such manner as to be effective and valid under applicable law. In the event a court of competent jurisdiction holds any provision of this Agreement to be invalid, such holding shall have no effect on the remaining provisions of this Agreement, and they shall continue in full force and effect.

30. Interp r e t a t i o n; Heading s . The word "including" shall mean "including without limitation". All pronouns and any variations thereof refer to the masculine, feminine or neuter, singular or plural, as the context may require. All terms defined in this Agreement in their singular or plural forms have correlative meanings when used herein in theirplural or singular forms, respectively. Headings used in this Agreement are for convenience of reference only and are not intended to influence the interpretation hereof.

31. G o v erning La w . This Agreement shall be governed by and construed in accordance with the domestic laws of the State of New York without giving effect to any choice or conflict of law provision or rule (whether of the State of New York or any otherjurisdiction) that would cause the application of the laws of any jurisdiction other than theState of New York.

32. N o S t r i c t Co nst r u c t i o n . The Parties have participated jointly in the negotiation and drafting of this Agreement. In the event of an ambiguity or question of intent or interpretation arises, this Agreement shall be construed as if drafted jointly by the Parties, and no presumption or burden of proof shall arise favoring or disfavoring any of the Parties by virtue of the authorship of any of the provisions of this Agreement.

33. Counte r pa r t s . This Agreement may be signed, including by facsimile signature, in two or more counterparts and each such counterpart will constitute an original document and such counterparts, taken together, will constitute the same instrument.

* * * * *

By:Name: Title:

Execution Copy

In witness to the foregoing, the Parties have executed this Research Agreement as of the date first written above.

FOUNDATION:

Institute of Animal Physiology andGenetics ASCR, v.v.i.

By: Name: Kopecny JanTitle: Director

The undersigned hereby agrees that I am the Researcher as defined in this Agreement. I also agree to be bound by the provisions of Section 6 and Section 7 and each of Section 14 and Section 15 of this Agreement to the extent that I am the "Receiving Party" of "Confidential Information" (both as defined in the Agreement). I hereby acknowledge that I have read this Agreement and further acknowledge that certain actions by me or my failure to perform certain actions shall constitute a breach of this Agreement by the Research Institution. I hereby assign, and agree to assign, to the Research Institution any and all right, title and interest I have in and to all my interest in the Patentable HD Invention and Other HD Intellectual Property.

RESEARCHER:

Zdenka Ellederova, Ph.D.

ResearchAgreernent_ CHDI_lnstAnima!PhysandGenetics_Ellederova_2016 _ReclD _A-11609ReclD: A-11609RevNo003 (040216)

Execution Copy

In witness to the foregoing, the Parties have executed this Research Agreement as of the date first written above.

FOllNDA TION:

CIIDI Foundation, Inc.

By: Name: Title:

Rl:st:ARCII INSTITUTION:

The undersigned hereby agrees that lam the Researcher as defined in this Agreemenl. I also agree to be bound by the provisions of Section 6 and Section 7 and each of Section 14 and Section 15 of this Agreement to the extent that I am the ''Receiving Pm1y"of"Confidcntial Information" (both as defined in the Agreement). I hereby acknowledge that I have read this Agreement and further acknowledge that certain actions by me or my failure to perform certain actions shall constitute a breach of this Agreement by the Research Institution. I hereby assign, and agree to assign, to the Research Institution any and all right, title and interest I have in and to all my interest in the Patentable HD Invention and Other HD Intellectual Property.

RESEARCHER:

Zdenka Lllederova. Ph.D.

ResearchAgreement_CHDl_lnstAnimalPhysandGenetics_Ellcdcrova_2016_RecID_A-11609Rec!D: A-11609RevNo003 (040216)

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ResearchAgreement_CHDI_InstAnimalPhysandGenetics_Ellederova_2016_RecID_A-11609RecID: A-11609

App e nd i x A T o R e s e ar c h Agre e m e nt

Foundation

Name: CHDI Foundation, Inc. Telephone: 212-660-8102

ContactPerson/Title:

Ruth Basu / ChiefAdministrative Officer Fax: 212-239-2101

Address: c/o CHDI Management, Inc.350 Seventh AvenueSuite 200New York, NY 10001

E-Mail: r uth . b a s u @ c h d i fo u n d a t i o n . o rg

Re s ea r ch I nstitution

Name: Institute of Animal Physiology and Genetics ASCR, v.v.i. Telephone: 00420 315 639 523

ContactPerson/Title:

Jan Kopecny, DSc.Director of The Institute ofAnimal Physiology and Genetics

Fax: +420 267 090 500

Address: The Czech Academy of SciencesRumburska 8927721 LibechovLibechov

E-Mail: k opecn yj@ i a p g . c a s . cz

AccountInformation: Komercni banka a.s.

Poboþka Melnik, Nam. Miru276 68 Melnik

SWIFT: KOMBCZPPXXXIBAN: CZ53 0100 0000 1982 6472 0227

Rese a r ch e r

Name: Zdenka Ellederova Telephone: +420 315 639 565

Title: Head of Laboratory of CellRegeneration and Plasticity

Fax: +420 315 639 510

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Address: Institute of Animal Physiology and Genetics ASCR, v.v.i. Rumburska 89277 21 LibechovCzech Republic

E-Mail: e ll e d e r o v a @ i a p g . c a s . cz

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R esea rch P roj ect – Tit le, Sta rt D ate a nd End Date

Research Project Title: Phenotypic Analyses of the HD Transgenic Minipig Model

Research Project Start Date: December 1, 2016

Research Project End Date: November 30, 2018

Rese a r ch P r o j ect – B u d get Summa r y (1)(2)

Local Currency CZK

Period 1 Period 2

12/01/16 - 11/30/17 12/01/17 - 11/30/18

I t em A m o u nt A m o u nt Personnel 1,155,375 1,155,375

Consumables 925,850 835,780

Animal Costs 0 0

Equipment(3) 0 0

Other Costs 0 0

Total Direct Costs for Period 2,081,225 1,991,155

Total Indirect/Overhead Costs for Period(4) 312,184 298,673

Total Budget for Period 2,393,409 2,289,828

Total Budget for Period (excluding Equipment) 2,393,409 2,289,828

Total Budget 4,683,237Total Budget (excluding Equipment) 4,683,237

(1) See the attached detailed budget description in respect of each budget period for detailed information relating to the budgeted line items.

(2) Provided that total expenditures do not exceed the total budget for the period, within a budget period the Researcher and the Research Institution may, without the prior written consent of the Foundation, expend up to 105% of the amount originally budgeted for an individual line item in the attached detailed budget. This does NOT apply to the following line items: Principal Investigator Personnel Cost, Equipment, Travel, and Indirect/Overhead Costs. Any line item expenditures exceeding 105% of the amount originally budgeted require prior written consent of the Foundation notwithstanding the fact that total expenditures do not exceed the total budget for the period.

(3) All purchased equipment shall be deemed "capital equipment" for purposes of S e cti on 1 7 ( f ) of this Agreement.

(4) Total Indirect/Overhead Costs are calculated as 15% (or such lower indirect/overhead rate applicable to the Research Institution) of the sum of total budgeted Personnel and Consumables costs. Total Indirect/Overhead Costs are subject to adjustment based upon total actual Personnel and Consumables expenditures; provided, however, the amount of Total Indirect/Overhead Costs payable by the Foundation in respect of a budget period will be the lower of (a) the original amount set forth in the Budget for such budget period and (b) 15% of the sum of the actual Personnel and Consumables costs for such budget period.


Personnel

N a m e Ti t le P r oject R o le

A n n u a l S al a r y

A n n u a l F r i nge

B e n e f its C o s t

A n n u a l F T E

A m ou n t L i n e I t e m

A m o u nt Taras Ardan MD, Ph.D researcher

Aim I599,000 30.00% 30.00% 233,610

Daniela Vidinska Mgr. Ph.D.studentAim I

294,000 30.00% 20.00% 76,440

Hana Rihova Technician TechnicianAim I-III

294,000 30.00% 50.00% 191,100

Hana Kovarova RNDr.CSc. researcherAim II

716,000 30.00% 10.00% 93,080

Ivona Valekova Mgr. Ph.D. Post docAim II

522,000 30.00% 20.00% 135,720

Stefan Juhás MVD. PhD researcher-Aim III

599,000 30.00% 20.00% 155,740

Matous Pokorný Ing. Ph.D. studentAim III

294,000 30.00% 20.00% 76,440

Monika Macáková MVD Ph.D.studentAim III

294,000 30.00% 20.00% 76,440

Zdenka Ellederová Ing. PhD PI,researcher in Aim I and Aim III

599,000 30.00% 15.00% 116,805

Total Personnel 1,155,375

Consumables

L i n e I t e m D e s c ri p t i o n L i n e I t e m

A m o u nt Tissue culture reagents (media, antibiotics, etc.) 27,900Molecular biology reagents 0Antibodies 275,890Chemicals/Pharmaceuticals 214,460Test Kits/Tools 115,000Supplies (surgical supplies; general lab supplies; plasticware; glassware; pipettes; filtering; personalprotection/sterility)

292,600

Total Consumables 925,850

Animal Costs

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Research Project – Detailed B udget – Bud get Period 1

Local12/01/16 - 11/30/17

Curre ncy CZK

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# C o st per L i n e I t e m T y p e S o ur c e A ni m als A n i m a l A m o u nt Acquisition No line items 0

T yp e Descr ipti onL i n e I t e m

Am ountHousing Animals 0Housing Cages 0

Li n e It emT yp e Li n e It em D escri pti on Am ountMiscellaneous No line items 0

Total Animal Costs 0

Equipment(1)

Li n e It em D escri pti onL i n e I t e m

Am ountNo line items 0

Total Equipment 0

Other Costs


Am ountNone 0

Total Other Costs 0

Total Direct Costs 2,081,225

Total Indirect/Overhead Costs(2) 312,184


(1) All purchased equipment shall be deemed "capital equipment" for purposes of S e cti on 1 7 ( f ) of this Agreement.(2) Total Indirect/Overhead Costs are calculated as 15% (or such lower indirect/overhead rate applicable to the Research Institution) of the sum of total budgeted Personnel and Consumables costs. Total Indirect/Overhead Costs are subject to adjustment based upon total actual Personnel and Consumables expenditures; provided, however, the amount of Total Indirect/Overhead Costs payable by the Foundation in respect of a budget period will be the lower of (a) the original amount set forth in the Budget for such budget period and (b) 15% of the sum of the actual Personnel and Consumables costs for such budget

peri o d.

Personnel

N a m e Ti t le P r oject R o le

A n n u a l S al a r y

A n n u a l F r i nge

B e n e f its C o s t

A n n u a l F T E

A m ou n t L i n e I t e m

A m o u nt Taras Ardan MD, PhD researcher

Aim I599,000 30.00% 30.00% 233,610

Daniela Vidinská Mgr. Ph.D. studentAim I

294,000 30.00% 20.00% 76,440

Hana Ríhová Technician TechnicianAim I-III

294,000 30.00% 50.00% 191,100

Hana Kovárová RNDr CSc. researcherAim II

716,000 30.00% 10.00% 93,080

Ivona Valeková Mgr.Ph.D. Post-docAimII

522,000 30.00% 20.00% 135,720

Stefan Juhás MVD PhD researcherAim III

599,000 30.00% 20.00% 155,740

Monika Macaková MVD Ph.D.studentAim III

294,000 30.00% 20.00% 76,440

Matous Pokorný Ing. Ph.D. AimIII

294,000 30.00% 20.00% 76,440

Zdenka Ellederova Ing. Ph.D PI,researcher Aim I and Aim III

599,000 30.00% 15.00% 116,805

Total Personnel 1,155,375

Consumables

L i n e I t e m D e s c ri p t i o n L i n e I t e m

A m o u nt Tissue culture reagents (media, antibiotics, etc.) 27,900Molecular biology reagents 0Antibodies 293,320Chemicals/Pharmaceuticals 219,060Test Kits/Tools 95,000Supplies (surgical supplies; general lab supplies; plasticware; glassware; pipettes; filtering; personalprotection/sterility)

200,500

Total Consumables 835,780

Animal Costs

T y p e S o ur c e #

A ni m als C o st per A n i m a l

L i n e I t e m A m o u nt

Acquisition No line items 0

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Research Project – Detailed B udget – Bud get Period 2

Local12/01/17 - 11/30/18

Curre ncy CZK

Execution


T yp e Descr ipti onL i n e I t e m

Am ountHousing Animals 0Housing Cages 0

Li n e It emT yp e Li n e It em D escri pti on Am ountMiscellaneous No line items 0

Total Animal Costs 0

Equipment(1)


Am ountNo line items 0

Total Equipment 0

Other Costs


Am ountNone 0

Total Other Costs 0

Total Direct Costs 1,991,155

Total Indirect/Overhead Costs(2) 298,673


(1) All purchased equipment shall be deemed "capital equipment" for purposes of S e cti on 1 7 ( f ) of this Agreement.(2) Total Indirect/Overhead Costs are calculated as 15% (or such lower indirect/overhead rate applicable to the Research Institution) of the sum of total budgeted Personnel and Consumables costs. Total Indirect/Overhead Costs are subject to adjustment based upon total actual Personnel and Consumables expenditures; provided, however, the amount of Total Indirect/Overhead Costs payable by the Foundation in respect of a budget period will be the lower of (a) the original amount set forth in the Budget for such budget period and (b) 15% of the sum of the actual Personnel and Consumables costs for such budget

peri o d.

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Research Project – Fixed Paym e n t s

Payment Number Payment Amount Date(s) and/or Condition(s) of Payment

Payment 1 CZK 797,803 Execution of this Agreement by the Parties.

Payment 2 CZK 797,803 Submission of an interim progress report in respect of theResearch Project activity during the period from12/01/16 through 05/31/17 reasonably acceptable to theFoundation.

Achievement of the milestones in respect of the period from 12/01/16 through 05/31/17 as set forth in the Description of the Research Project attached hereto as A pp en d i x B to the reasonable satisfaction of the Foundation.

Payment 3 CZK 797,803 Submission of an interim progress report in respect of theResearch Project activity during the period from06/01/17 through 11/30/17 reasonably acceptable to theFoundation.


Submission of a financial audit report in respect of the budget period beginning 12/01/16 and ending 11/30/17 reasonably acceptable to the Foundation.

Payment 4 CZK 763,276 The Foundation has not given notice to the Research Institution pursuant to S ec ti o n 2 ( c ) of this Agreement that the Foundation has determined not to provide financial support for Budget Period 2 of the Research Project.


Submission of an interim progress report in respect of theResearch Project activity during the period from12/01/17 through 05/31/18 reasonably acceptable to theFoundation.

Achievement of the milestones in respect of the period from 12/01/17 through 05/31/18 as set forth in the Description of the Research Project attached hereto as


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App e n d ix B to the reasonable satisfaction of theFoundation.


Submission of a final progress report in respect of theResearch Project activity during the period from06/01/18 through 11/30/18 reasonably acceptable to theFoundation.


Submission of a financial audit report in respect of the budget period beginning 12/01/17 and ending 11/30/18 reasonably acceptable to the Foundation.

Total Payments CZK 4,683,237

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App e nd i x B t o R e s e ar c h Agre e m e nt

(Description of Research Project)


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P he not yp ic Ana lys e s o f t he HD Tr a ns ge nic M inip ig Mode l

SUMMARY

The transgenic model of Huntington's disease in minipig (TgHD) was created in 2009 (Baxa et al2013) and information coding the sequence of N-terminal part of human mutated huntingtin wastransferred to subsequent generations from both female and male sides. In each litter, transgenic (TgHD) and wild-type (WT) piglets were born in approximately equal ratio. At present, the Laboratory of Cell Regeneration and Plasticity keeps sets of animals in F1, F2 and F3 generations with identical genetic background and bred in identical conditions of feeding and housing. The presented project outlines a prolongation of the research agreement focused on a complex of non-invasive and invasive approaches to WT and TgHD minipigs to achieve the entire phenotypic analysis of HD progression in this large animal model.

Lately we established behavioral tests on our oldest animals in order to detect the first behavioral and motoric phenotype in TgHD (Sp.aim III). Furthermore, we plan to focus on the quantitative cytokine/chemokine profiling in cerebrospinal fluid, blood serum and peripheral blood monocytes/macrophages secretome in F2 generation of minipigs (Sp. aim II). Cell lines of skin fibroblasts isolated from WT and TgHD minipigs (F0,F1) will be measured for mtHtt expression and its protein conformers, fibrillar oligomers and aggregates (Sp. Aim I).

Moreover, invasive approach will be applied in F2 generation of minipigs and selected cytokine/chemokine levels in primary microglia secretome will be assessed (Sp. aim II). Oligomers, monomers and fragments of mtHtt will be investigated in tissue lysates from 48 and60 months old animals, special attention will be paid to characterization of the brain health statusand HD markers in 48 and 60 months old TgHD minipigs by using histological and immunohistochemical (IHC) approach (Sp. aim I). In conclusion, the proposed experiments included in Sp. aims I-III will produce complex information about preclinical stages of HD in transgenic minipigs in relevance to human patients.

Background and Preliminary Data

Aim 1. Biochemical and histological studies

HD manifestation is characterized by accumulation and aggregation of mtHtt primarily in striatum and cortex. The presence of large inclusion bodies, resp. aggregates, is tightly correlated with progression of HD (DiFiglia et al. 1997). However, recent studies suggest protective role of aggregates against the effects of mtHtt (Arrasate et al. 2004). On the contrary, soluble monomers of mtHtt and huntingtin oligomers were described to be toxic to cells and to be the key factors of cellular dysfunction (Lajoie & Snapp 2010). In affected tissues, normal huntingtin is present in intact soluble monomeric form but mtHtt is found in fragmented, oligomerized and polymerized forms (Hoffner et al. 2007).


Execution We investigate aging animals for presence of aggregates by various methods that are able to reveal mtHtt aggregates. We have detected just a few aggregates in 24 and 36 months old sections by immunohistochemistry, almost nothing compare to the R6/2 mice sections (Fig. 1).

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However, filter retardation assay showed retarded Htt polymers in cortexes of TgHD minipigs compare to wild type (WT) (Fig. 2), but not in other tested tissue or cells.

Furthermore, we have detected oligomeric smears using anti Htt antibody EPR 5526 in the brain and testes samples of 24 and 36 months old TgHD minipig and not in the samples of wild type (WT) siblings (Fig. 3,4). In addition, we could not detect the oligomeric smears in other tissues (Fig.4).

Next, we introduced equilibrium sedimentation method (Laferriere et al. 2013) from colleges from Magda Polymenidou's group in Zurich studying Amyotrophic lateral sclerosis by this method. This is another approach for detection higher insoluble structures (see the first RA six months report of 2015).

Further, Miller et al. (2011) showed toxic monomers and possibly small oligomers containing expanded polyQ are recognized by 3B5H10 antibody (Miller et al. 2011). Surprisingly, 3B5H10 monoclonal antibody detected several lower molecular weight fragments of Htt in our TgHD minipig brain tissues, but none in the brain tissue from wild type siblings (Fig.5). Interestingly, muscles from 24 months old animals did not show any fragmented mtHtt, and muscles form 36 months old animals showed just minor fragmentation compared to the mtHtt fragments in testes (Fig. 5C).

We suggest that relatively high levels of all proteolytic enzymes detected in TgHD minipig can augment production of mtHtt derived proteolytic fragments and thus contribute to the disease development. Especially the increased expressions of matrix metalloproteinase-9, caspase-3, and partly calpain-5 in comparison to WT animals suggest a more intense cleavage of mtHtt into toxic small fragments (Fig.6). Due to different expression of proteolytic enzymes in TgHD brains further studies on older TgHD minipigs (48m, 60m) are necessary to clarify the exact role of these enzymes in the HD etiology. Because of mtHtt affects within the brain not only striatum, but also cortex, we will also evaluate selected cortical areas (motor, somatosensory and insular cortex), which are often associated with HD. In this way we will be able to utilize all obtained information from staining coronal section and monitor additional important areas in which processes associated with mtHtt presence get underway.

Although we detected mostly minor changes in expression of selected HD markers (slightly increased expression of IBA1 and GFAP; decreased DARPP32) in 36 months old TgHD minipigs we expect the strengthening of these changes in 48 and 60 months old animals (Fig. 7).

Furthermore, significantly decreased myelination of nerve fibers of striatum was detected in 36 months old TgHD minipig (K63) in comparison to WT (K64) (it was seen in progressive report for the first year). We plan histochemical examination with Toluidine blue and Luxol fast blue in coronal brain sections detecting changes in cellularity and myelinization.

Since invasive approaches in large animal models need to be limited, non-invasive approaches are preferred. We successfully cultivate skin fibroblasts and detect Htt in them. Therefore we will continue to detect expression and different forms of mtHtt also in cells.

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Investigation of different forms of mtHtt, proteolytic enzymes as well as HD markers is necessary for following the progression of disease in our transgenic minipig model and thus helping describe the model for preclinical testing of potential therapies for Huntington's disease.

Aim 2. Immuno-inflammatory studies in TgHD mini-pigs

It is believed that the primary pathology of HD results from massive degeneration of neurons in the basal ganglia. However, the expression of mutant huntingtin was detected in all examined tissues. Thus the mutation in non-neuronal cells both within the brain and in peripheral tissues contributes to the pathology of HD (Moller 2010; Ellrichmann et al. 2013). Neuroinflammation, especially microglia activation, is well characterized feature of neurodegenerative diseases. Microglia appear in the brain during inflammation and expand rapidly locally causing microgliosis (Ajami et al. 2007) . Furthermore, post mortem study of HD brains has shown that the presence of reactive microglia was significantly identified in regions most damaged in HD, notably striatum and cortex (Sapp et al. 2001). In vivo PET study supported these findings by indicating that microglia activation appears in the brains of pre-symptomatic HD subjects (Tai et al. 2007). Other studies have shown that neuroinflammation is also caused by release of pro- inflammatory cytokines and chemokines from the microglial cells.

Elevated levels of inflammatory cytokines and chemokines in HD human plasma samples correlate with disease severity and are detected many years before the onset of motor symptoms (Bjorkqvist et al. 2008; Wild et al. 2011). Several studies provided evidence that non-neuronal cells might play important role in HD progression and behave abnormally. Circulating peripheral blood monocytes from HD subjects expressed mutant huntingtin and were pathologically hyperactive in response to stimulation with LPS (Bjorkqvist et al. 2008; Crotti & Glass 2015; Trager et al. 2015).

We assume that mutant huntingtin induces immune alterations in the central nervous system as well as in the periphery in our transgenic minipig model. In our previous work we identified IFNĮ and IL-10 proteins as promising biomarkers in CNS (Fig. 8, Fig. 9) and IL-8 in blood serum (Fig. 10) reflecting immune-pathological mechanisms in porcine HD model at the age of36 months (Valekova et al. 2016).

Aim 3. Behavioral studies

Huntington's disease is characterized by involuntary chorea like movements, poor balance, cognitive dysfunction, and psychiatric problems manifesting in the middle age of the patients. Animal models, for example R6/2 mice also exhibit difficulties in a number of tasks namely swimming, beam traversing, and maintaining balance on the Rota-rod at the fastest rotating speeds. EEG, ECG, activity and core temperature monitoring were successfully measured in HD mouse models (R6/2 and R6/1) by telemetry devices (Kiriazis et al. 2012; Fisher et al. 2013). Deficits in sleep and circadian organization have been identified as common early features in patients with HD. They correlate with severity of the symptoms and may be instrumental in disease progression.

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We have established several behavioral tests in our TgHD minipig model in order to follow any changes concerning behavioral, motoric, or cognitive impairment in our facility. The recently

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realized set of behavioral tests in F0 and F1 WT and TgHD minipigs (around 6 to 7 years old) indicated phenotype development in motoric and behavioral level (differences in walking and turns, stability test, balance beam, video 1-3 in supplements).

Toy 1 cover pan

This test was used to test the pig's ability to learn. First, the biscuit was inserted into an open hole. Then it was put in a half closed hole and we monitored whether the pig was able to open the hole to get the biscuit.

Toy 2 skittles

Also this test was used to test the pig's ability to learn. Biscuits were hidden under the skittles. And we tested weather the pig was able to actively uncover the skittles.

Hurdle

We evaluated the ability of the pig to cross a hurdle. In addition to fearlessness to cross the barrier, we also evaluated the coordination during crossing.

Tunnel test

We put three biscuits in to the tunnel in different distances. And we counted how many biscuits could the pig get evaluating the fearlessness of the pig to enter the tunnel.

Seesaw

This test was evaluating the pig's balance.

The results of the preliminary studies using 15 pigs, 8 TgHD, and 7WT of F0, F1, and F2 generations at the age of 4 to 6 years aresummarized in Fig. 11. This data showed some motor and behavioral changes in TgHD animalscompared to WT. We observed reduced fearlessness, problems with seesaw test, which may involve an impaired locomotor coordination. Above all, we observed strange laying of legs while walking in some TgHD animals from all three generations but not in WT minipigs (See video 1 in supplements). This could be a possible start of physical changes characteristic for HD.

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Furthermore, we built constructed an elevated balance beam in order to increase a stress situation for the pigs. This approach was recently published in porcine model with ataxia telangiectasia (Beraldi et al. 2015). For this case, we divided the group of 15 previously tested animals into F0- F1 and F2 generations. In the older group (F0-F1), 5-6 years old, we saw a significant delay in

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learning to cross the balance beam (Fig. 12) (Fig.13, also see video 2 in supplements). In comparison, the younger group (F2), 4.5years old, did not yet show any differences between WT and TgHD animals (Fig. 14). Moreover, we introduced a stability test (the pull test), where we detected a significant difference in TgHD vs WT boars of F1 generation (video 3 in supplements). The pull test is commonly used as a measure of postural instability (reactive balance impairment) in patients with movement disorders. The patient stands with feet comfortably apart with opened eyes. Examiner stands behind close to the patient and quick pull backward by the shoulders to see how the patient compensates for the sudden imbalance.

Recently, we purchased a telemetric system rodentPACK2 and established telemetric measurements of the TgHD minipigs. RodentPACK2 has been primarily designed for rodent studies. This fact has forced us to newly develop prototype equipment interconnecting connectors screwed to external transmitters with internal electrodes into the minipig´s body (Fig.15). We developed an automatic system for measurement and evaluation of their physical activity (Pokorny et al. 2015).

Göttingen minipigs (Sus scrofa domesticus) represent an ideal large animal model for biomedical studies due to their relatively small size, characterized health status, and ease of training and handling (Willens et al. 2014). Moreover, Yorkshire pigs were also used in long-term, simultaneous telemetric monitoring of blood flow, pressure and heart rate in heart failure models(Choy et al. 2014). EEG, ECG, activity and core temperature monitoring were successfully measured in HD mouse models (R6/2 and R6/1) by telemetry devices (Kiriazis et al.2012; Fisher et al. 2013). Deficits in sleep and circadian organization have been identified as common early features in patients with HD. They correlate with severity of the symptoms and may be instrumental in disease progression. These observations were also detected in the most employed mouse model (R6/2) (Fisher et al. 2013) or ovine model of HD (Morton et al. 2014). Our preliminary data on 3 years old animals showed a significant decrease of total acceleration representing physical activity in TgHD minipigs between 4:40 and 5:30 a.m. (after night sleep – before morning feeding) in comparison with WTs (Fig.13). This could be explained by a disturbed energy metabolism. The telemetry approach could contribute to the deeper characterization of HD minipig model in terms of physical activity in their preclinical and clinical phase.

Specific Aims/Goals

Aim 1. Biochemical and histological studies

G o a l s :

x To investigate and semiquantify mutant and endogenous Htt expression and its forms in tissues (mainly brains) of TgHD minipigs (F2 generation, 48 and 60 months old)

x To localized aggregates presence in brains of TgHD minipigs (F2 generation, 48 and 60 months old)

x To characterize and semiquantify expression of markers for glial activation in brains of

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TgHD minipigs (F2 generation, 48 and 60 months old)

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x To perform immunohistochemical analysis of proteolytic enzymes expression in brains ofTgHD minipigs (F2 generation, 48 and 60 months old)

Aim 2. Immuno-inflammatory studies in TgHD mini-pigs

Go a l s

x Quantitative analysis of selected cytokine/chemokine levels in peripheral blood monocytes and macrophages secretome (F2 generation, age of 48 – 56 months)

x Quantitative analysis of selected cytokine/chemokine levels in primary microglia secretome (F2 generation, age of 48 months)

x Quantitative analysis of cytokine/chemokine levels of blood samples and cerebrospinal fluid (F2 generation, age of 48 months; F3 generation, age of 42 and 48 months)

x Comparison of sample proteomes (microglia vs monocytes, CSF vs serum) obtained from transgenic porcine model of HD and their wild-type counterparts

Aim 3. Behavioral studies

Go als

x To follow behavioral, motoric, and cognitive changes in the oldest animals 5-8 years old, F0, F1 and F2 generations of transgenic minipigs (TgHD) and compare them with their wildtype (WT) siblings.

x To measure the physical activity disturbances characterized by deficit in sleep and circadian organization of TgHD in comparison with wild type (WT) minipigs by telemetry. F3 generation, 4– 6 years old animals will be used.

Experimental Plan/Description of Research Project Activities

Desc r i p t i o n o f R e sear c h P r o j ect A c t iv i t ies

Aim 1:

Samples: During the second year of RA, we plan to investigate wild type (WT) and transgenic(TgHD) minipigs of F2 generation (48 and 60 months old).

Our goal for this year proposal is to continue to monitor and describe our minipig model of HD. We will follow expression and accumulation of mtHtt, and the presence of fragmented, oligomerized and polymerized forms of Htt. Furthermore we would like to focus on cytoplasmic and nuclear localization and phosphorylation of Htt and detection of HD markers.

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The oldest animals up to date are six and half (F0) and six (F1) years old. Recently, we sacrificed three pairs of 48 months old animals, and froze tissues for further experiments. We plan to


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sacrifice three pairs of 60 months old animals in summer 2016. Among the frozen tissues we will mainly focus on brain: striatum (caudate nucleus and putamen) and cortex. Afterwards spinal cord, heart, testes, and muscle tissues will also be examined. In the meanwhile we will use noninvasive approaches in our F0 and F1 generations of TgHD and WT minipigs using skin fibroblasts. All experiments will be done in triplicates.

Expression of Htt will be investigated at the RNA level as well as protein level. i) qPCR will be performed ii) western blot with Htt specific antibodies such as EPR5526, MAB2166 verified in our lab, will be used, and results will be evaluated using quantification software. Huntingtin is present mainly in cytoplasm but a small amount can be found in nucleus. The amount of Htt in nucleus is increased, if the cell undergoes stress, and also in certain points of cell cycle (Atwal et al. 2011). To elucidate whether localization of Htt in TgHD minipigs is altered, we will separate the nuclear and cytoplasmic fractions before analysis. Longitudinal study should reveal whether endogenous or transgenic Htt accumulates in brain, skin fibroblast and buccal mucosa, and if it changes its localization. Immune-/histochemical methods and image analysis techniques will be employed for qualitative detection and semi-quantitative determination of protein expression in the brain tissue. Sections will be stained mainly with antibodies for the detection of huntingtin (WT/mtHtt) (EPR5526, BML-PW0595), Htt large aggregated form (MW8). We also plan to use a new antibody for aggregates from Ali Khoshnan. HD markers detected in this study will include: glial markers (GFAP, IBA1) and medium sized striatal neurons (DARPP-32), or staining with Toluidine blue and with Luxol Fast Blue for myelin detection.

Because of mtHtt affects in the brain not only striatum (even if mainly) but also cortex, we will also evaluate selected cortical areas (motor, somatosensory and insular cortex) which are often associated with HD. In this way we will be able to utilize all obtained information from staining coronal section and monitor additional important areas in which processes associated with mtHtt presence get underway. The specificity of the primary antibodies will be verified by Western Blot and in some cases also by comparative immunohistochemistry (IHC) of WT and TgHD mouse (R6/2, 12 weeks old) brain sections. The control specificity of secondary antibody will be proved by omission of primary antibody from the incubation media.

Next, we will test samples for the presence of aggregates. Aggregates will be detected using various approaches. i) Immunohistochemistry – see above. ii) Seprion bead capture (Microsens Biotechnologies) Cell or tissue lysates will be exposed to the beads, beads will be washed and samples loaded on SDS-PAGE gel, and western blot with anti-Htt antibodies will be performed. iii) Filter retardation assay - based on the fact that very large polymers cannot pass through 0.2µm cellulose acetate filters and therefore can be isolated. iv) Velocity sedimentation method(Laferriere et al. 2013) on linear optiprep gradient (10-25%) with two solubilization steps using ultracentrifuge at 55 000 rpm, Formic acid treatment can dissociate non-covalently stabilized aggregates otherwise unable to enter the gel. The assay will be performed as described (Scherzinger et al. 1997). In all methods R6/2 mouse brain samples will be used as positive control. In progression of the disease we expect to detect aggregates in certain age of TgHD minipigs.


Execution Oligomers and monomers will be investigated mainly by Western blot, taking advantage of oligomer retardation in stacking gel and using oligomer specific antibodies, for example:

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EPR5526, MW8 - a commercial antibody against N-terminal end recognizing alpha-helical, random coil, and extended conformations of huntingtin. In order to distinguish between soluble and insoluble forms stabilized by covalent bonds, formic acid, which dissolves non-covalent but not covalent bonds, will be used. We have already detected oligomer forms in 24 and 36 months old brain tissues (see preliminary data) and testes (data not shown). Therefore we expect to find oligomers also in 48 and 60 months old brains. We are interested in whether oligomers will be also present in various tissues as well as in skin fibroblasts and buccal mucosa swabs as the minipigs age.

Fragment detection. Since 3B5H10 (a commercial antibody raised against an N-terminal 171 amino acid fragment of Htt with 65Q), 1C2, and Ab1 antibodies reveal several lower bands (see preliminary data), we will perform western blots with these antibodies to examine tissues, skin fibroblasts and buccal mucosa in different ages of the animals. To determine whether fragments are present in cell nucleus, cytoplasmic and nuclear fractions will be separated (see preliminary data). We expect to see differences in the amount of fragments in different tissues, skin fibroblasts and buccal mucosa swabs of TgHD compared to WT and their accumulation with age of the animals. To this end we also plan immunostaining the brain sections with antibodies anti proteolytic enzymes (MMP9, MMP10, CPN5, KLK10), which participate on toxic mtHtt fragmentation. The results will be verified by Western blotting.

Phosphorylation on N17 It is known from literature that dephosphorylated Ser 14 and Ser 16 of Htt leads to malformation of Htt. Mutated huntingtin hypophosphorylated on N17 is cytotoxic. The mtHtt in minipigs seems to be phosphorylated in the present age of animals (data not shown). Therefore we will continue monitoring its phosphorylation in older animals. We will perform western blots and immunohistochemistry with anti-Pi-N17, a kind gift from Dr. Ray Truant. We expect dephosphorylation of Ser 14 and Ser 16 in certain age of animals.

Aim 2:

Samples: We intend to study following samples:

- Peripheral blood monocytes 6 pairs

- Macrophages differentiated from monocytes 6 pairs

- Primary microglia from cerebellum 2 – 4 pairs

- Cerebrospinal fluid 6 pairs (F2) + 2x 6 pairs (F3)

- Blood serum 6 pairs (F2) + 2x 6 pairs (F3)

Isolation of peripheral blood monocytes, immune phenotyping and stimulation

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Whole blood will be obtained from littermate minipigs (48 – 56 months – 6 TgHD and 6 WT). Peripheral blood mononuclear cells (PBMCs) will be isolated by density gradient centrifugation

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over Ficoll-Paque Plus 1077 solution and monocytes will be obtained from PBMCs suspension by magnetic sorting using anti-CD14 microbeads (Miltenyi Biotec). The purity and immune phenotyping of obtained monocytes will be determined by flow cytometry using two-color staining the cells for CD14 and CD3 antibodies. Stimulation will be done using 10 units/ml IFNȖ in combination with or without LPS (3 µg/ml) for 24 h. Cell culture supernatants will be collected, centrifuged and stored at -80°C for further analysis.

Differentiation of primary isolated monocytes to macrophages, immune phenotyping and stimulation

CD14+ monocytes (48 – 56 months – 6 TgHD and 6 WT) will be isolated from whole blood, as previously described, and differentiated into macrophages by supplementing the culture medium with 20 ng/ml recombinant porcine GM-CSF for 6 days. The purity and immune phenotyping of obtained macrophages will be determined by flow cytometry using CD14 and CD163 antibodies. Stimulation will be done using 10 units/ml IFNȖ in combination with or without LPS (10 and100 ng/ml) for 24 h. Cell culture supernatants will be collected, centrifuged and stored at -80°Cfor further analysis.

Isolation of primary microglia from minipig cerebellum, immune phenotyping and stimulation

Littermate TgHD and WT minipigs (48 months – 2 - 4 TgHD and 2 - 4 WT) will be used for isolation of primary microglia. The isolation of microglial cells from cerebellum will be performed using papain dissociation kit (Papain Dissociation System, Worthington Biochemical Corp) followed by a discontinuous density gradient centrifugation using Percoll. Microglial cell purity and immune phenotype will be determined by immunofluorescence and flow cytometry analyses of known microglial markers. For immunofluorescence staining IBA-1 protein as a marker of microglial cells will be chosen. Flow cytometry analysis will be undertaken to analyze immune-staining of surface CD molecules typically expressed by microglia including CD45, CD14 and CD172a. Microglial cells will be stimulated using 10 units/mL IFNȖ in combination with LPS (10 or 100 ng/mL) for 8 h and 24 h. Cell culture supernatants from stimulated and non- stimulated cells will be collected, centrifuged, and stored at -80°C for further analysis.

Cerebrospinal fluid and blood serum collection

The TgHD minipigs as well as their WT siblings at age of 48 months will be included in this part of study (F2 generation – 6 TgHD and 6 WT). Furthermore, CSF and serum will be collected from 42 and 48 months old minipigs (F3 generation – 2x 6 TgHD and 6 WT).

Luminex xMAP multiplex analysis of cytokines

All samples (blood serum, CSF, monocytes, macrophages and microglia secretomes) will be analyzed using Swine Cytokine Magnetic 7-plex Panel (Life Technologies) enabling the

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simultaneous measurement of IL-1ȕ, IL-4, IL-8, IL-10, IFNĮ, IFNȖ, and TNFĮ. This panel works according to our previous tests very well for this type of samples. Only for CSF measurements, we will need to modify the manufacturer's protocol, as the differences in protein level between CSF and serum are too high (ca. 200x lower total protein concentration in CSF) and this might affect the assay performance. We plan to use Luminex xMAP assays for the analysis of CSF and

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complementary blood serum samples. The measurements will be performed on Luminex 200 instrument (Luminex Corp., USA). Obtained data sets will be analysed in collaboration with Thermo Fisher Scientific, USA. We will use a precise weighted 5-parameter equation for cytokine quantification. In comparison to the original xPonent software (Luminex Corp., USA), we will be able to quantify significantly more samples, mostly in the lower concentration range. Statistical analysis will be performed using Instat 3 software (GraphPad Software, USA).

Aim 3:

All experiments will be conducted with the approval of the State Veterinary Administration of the Czech Republic and in accordance with Czech regulations and guidelines for animal welfare.

a) Behavioral tests on already established group of animals from F0, F1 and F2 generations. The minpigs will be evaluated within two groups. The younger group, F2 generation, will consist of five WT and five TgHD animals. The older group, F0 and F1 generations, will consist of 3 WT and 3TgHD. All animals will be weighted every two months

We will train the animals using kicker and biscuits. We will perform above in details described behavioral tests such as Hurdle test, Tunnel test, Seesaw test, Toy 1 cover pan, Toy 2 skittles, Balance beam, and pull back stability test. We will have a scale of marks for each test, where 5 will be the best performance and 0 the worst. The results will be statistically evaluated using GraphPad PRISM software (GraphPad Software, San Diego, CA, USA).

Furthermore, new methods and approaches will be developed in order to deeper follow the behavioral and motoric phenotype of the TgHDminipigs, such as step down.

b) For the telemetric measurements we will use F3 generation comparing TgHD (N = 6) and control wild type siblings (N = 6). All of the animals will be boars. The boars will be individually held in the neighboring boxes (one group 3TgHD + 3WT in one part of the stable and the second one 3TgHD + 3WT in another part of the stable).

We will use recently established telemetric system rodentPACK2 obtained from Emka TECHNOLOGIES (France). For this study we will use the established measurement of acceleration channels. Data will be acquired at sampling frequency 100 Hz with resolution ± 2 g during night and day. All events during the measurement session will be recorded e.g. time of feeding, cleaning, veterinary and zoo technician intervention or other activities. Boars will wear the empty collars, even when the experiment will not be running to eliminate the collar influence on their behavior.

Furthermore, we will also test a new type of mounting approach of the system to the minipig to be able to detect next to the acceleration also biopotentials (EEG, ECG, EEG, body temperature) of the minipigs. We have already performed some minipig pilot analysis of biopotentials, but they were complicated with secondary infection from the mounting of internal electrodes into the minipig´s body.

If the new type of mounting will be functioning in first 2 animals, we could implant devices for all animals (6 TgHD + 6 WT).

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For the statistical analysis we use two softwares provided by Emka TECHNOLOGIES – iox2 for data acquisition and real-time analysis as well as ecgAUTO for complex analysis. Total acceleration (gravity acceleration - m/s2) representing physical activity of animal will be processed in ecgAUTO software. The total physical activity will be sampled at 100 Hz and averaged over 10 minutes. Generated physical activity (mean from 10 minutes) from five TgHD and five WT boars will be consequently averaged and used for statistical analysis. A one-way ANOVA test with Bonferroni's multiple comparison post-test will be employed using GraphPad PRISM software (GraphPad Software, San Diego, CA, USA).

Founda t i o n Pr o v i ded Ma t e r i a l s

MW8 (CHDI-90000942): 14 vials (100uL/vial at 3.65 mg/mL), a monclonal antibody to Htt exon-1 with 67Q fused to glutathione-S-transferase but boosted with Htt exon-1 with 67Q (aggregate) that produces anti huntingtin monoclonal antibody.

The Foundation shall provide to the Research Institution up to 16 pigs containing N-Terminal truncated (548aa) mutant human Huntingtin with 125 PolyQ Repeats (CHDI-84001001, such pigs shall hereinafter be referred to as the "F ra g me n t P i g s ") aged 48 to 60 months old for use in accomplishing aim 1 activities, up to 25 Fragment Pigs aged 42 to 56 months old for use in accomplishing aim 2 activities, and up to 17 Fragment Pigs aged 5 to 9 years old for use in accomplishing aim 3 activities. The Research Institution is already in possession of such Fragment Pigs pursuant to that certain Pig Breeding and Maintenance Services Agreement, dated as of April, 1, 2012 (the "Services A gre e m e n t "), and that certain Supplement No. 01 to the Services Agreement, dated as of April 1, 2012. During the term of the Research Agreement, the Foundation shall continue to provide financial support to the Research Institution for maintenance of the Fragment Pigs pursuant to the terms of the Services Agreement. Upon completion of the Research Project, any remaining Fragment Pigs shall continue to be subject to the terms of the Services Agreement.

Milestones

Aim 1:

S ix- Mo nt h M ile st o nes

x Qualitative and quantitative characterization of different forms of mtHtt including monomers, oligomers and aggregates, and functional markers for medium sized striatal neurons and glial cells (microglia and astrocytes) as well as Toluidine blue and Luxol fast blue staining of coronal sections from 48 months old animals using biochemical and immunohistochemical methods.

Tw e l v e-M o n t h Miles t o n es

x Qualitative and quantitative characterization of proteolytic enzymes from the group of matrix metalloproteinases, calpains and kallikreins, and additional markers in coronal

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sections from 48 months old animals using biochemical and immunohistochemical methods.

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E i g h t e e n - M o n t h M il e st o n e s

x Qualitative and quantitative characterization of different forms of mtHtt including monomers, oligomers and aggregates, and functional markers for for medium sized striatal neurons and glial cells (microglia and astrocytes) as well as Toluidine blue and Luxol fast blue staining of coronal sections from 60 months old animals using biochemical and immunohistochemical methods.

T went y- fo ur- Mo nt h Mile st o nes

x Qualitative and quantitative characterization of proteolytic enzymes from the group of matrix metalloproteinases, calpains and kallikreins, and additional markers in coronal sections from 60 months old animals using biochemical and immunohistochemical methods.

Aim 2:

S ix - M o n t h M il e s t o nes

x Non-invasive approaches

Isolation of peripheral blood monocytes, immune phenotyping and stimulation

Differentiation of primary isolated monocytes to macrophages, immune phenotyping and stimulation

Production and secretion of cytokines by isolated and in vitro cultured monocyte/macrophage secretomes using xMAP technology

Tw e l v e-M o n t h Miles t o n es


Cerebrospinal fluid collection (F2

generation) Blood serum collection (F2

generation)

Quantitative analysis of cytokine levels of cerebrospinal fluid (F2

generation) Quantitative analysis of cytokine levels of blood samples (F2

generation)

E i g h t e e n - M o n t h M il e st o n e s

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Cerebrospinal fluid collection (F3 generation)

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Blood serum collection (F3 generation)

Quantitative analysis of cytokine levels of cerebrospinal fluid (F3

generation) Quantitative analysis of cytokine levels of blood samples (F3

generation)

T went y - f o ur M o n t h Mil e s to nes

x Invasive approaches

Isolation of primary microglia from minipig cerebellum, immune phenotyping and stimulation

Production and secretion of cytokines by isolated and in vitro cultured microglia secretomes using xMAP technology

Comparison of sample proteomes (microglia vs monocytes/macrophages, CSF vs serum)obtained from transgenic porcine model of HD and their wild-type counterparts

Aim 3:

S ix - M o n t h M il e s t o nes

x Testing and evaluating two groups (F0-F1-born 2009-2010, and F2 born 2011) at the age of 5 and 6 to 7 years using above described behavioral tests. Establishing also a few new tests and approaches, which could be relevant to follow the behavioral phenotype in extend.

x Measuring the physical activity disturbances by telemetric testing of 3.5 years old TgHD and WT (F3 generation) animals. Data analysis of telemetric physical activity measurements.

Twe lve-Mo nt h Milest o nes

x Testing and evaluating two groups (F0-F1-born 2009-2010, and F2 born 2011) at the age of 5.5 and 6.5 to 7.5 years using above described tests and some other new established behavioral tests.

x Data analysis of telemetric physical activity

measurements. E i g h t e e n - M o n t h M il e st o n e s

x Testing and evaluating two groups (F0-F1-born 2009-2010, and F2 born 2011) at the age of 6 and 7 to 8 years using above described behavioral tests. Establishing also a few

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new tests and approaches, which could be relevant to follow the behavioral phenotype in extend.

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x Measuring the physical activity disturbances by telemetric testing of 3.5 years old TgHD and WT (F3 generation) animals. Data analysis of telemetric physical activity measurements.

T we nt y - f o u r- M o n t h M il e st o n e s

x Testing and evaluating two groups (F0-F1-born 2009-2010, and F2 born 2011) at the age of 6.5 and 7.5 to 8.5 years using above described tests and some other new established behavioral tests.

x Data analysis of telemetric physical activity measurements.

Referen ces

Ajami B., Bennett J.L., Krieger C., Tetzlaff W. & Rossi F.M. (2007) Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci 10,1538-43.

Arrasate M., Mitra S., Schweitzer E.S., Segal M.R. & Finkbeiner S. (2004) Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431,805-10.

Atwal R.S., Desmond C.R., Caron N., Maiuri T., Xia J., Sipione S. & Truant R. (2011) Kinase inhibitors modulate huntingtin cell localization and toxicity. Nat Chem Biol7, 453-60.

Baxa M., Hruska-Plochan M., Juhas S., Vodicka P., Pavlok A., Juhasova J., Miyanohara A., Nejime T., Klima J., Macakova M., Marsala S., Weiss A., Kubickova S., Musilova P., Vrtel R., Sontag E.M., Thompson L.M., Schier J., Hansikova H., Howland D.S., Cattaneo E., DiFiglia M., Marsala M. & Motlik J. (2013) A transgenic minipig model of Huntington's Disease. J Huntingtons Dis 2, 47-68.

Beraldi R., Chan C.H., Rogers C.S., Kovacs A.D., Meyerholz D.K., Trantzas C., Lambertz A.M., Darbro B.W., Weber K.L., White K.A., Rheeden R.V., Kruer M.C., Dacken B.A., Wang X.J., Davis B.T., Rohret J.A., Struzynski J.T., Rohret F.A., Weimer J.M. & Pearce D.A. (2015) A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease. Hum Mol Genet 24, 6473-84.

Bjorkqvist M., Wild E.J., Thiele J., Silvestroni A., Andre R., Lahiri N., Raibon E., Lee R.V., Benn C.L., Soulet D., Magnusson A., Woodman B., Landles C., Pouladi M.A., Hayden M.R., Khalili-Shirazi A., Lowdell M.W., Brundin P., Bates G.P., Leavitt B.R., Moller T.& Tabrizi S.J. (2008) A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease. J Exp Med 205, 1869-77.

Crotti A. & Glass C.K. (2015) The choreography of neuroinflammation in Huntington's disease.Trends Immunol 36, 364-73.

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DiFiglia M., Sapp E., Chase K.O., Davies S.W., Bates G.P., Vonsattel J.P. & Aronin N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277, 1990-3.

Ellrichmann G., Reick C., Saft C. & Linker R.A. (2013) The role of the immune system inHuntington's disease. Clin Dev Immunol 2013, 541259.

Fisher S.P., Black S.W., Schwartz M.D., Wilk A.J., Chen T.M., Lincoln W.U., Liu H.W., Kilduff T.S. & Morairty S.R. (2013) Longitudinal analysis of the electroencephalogram and sleep phenotype in the R6/2 mouse model of Huntington's disease. Brain 136, 2159-72.

Hoffner G., Soues S. & Djian P. (2007) Aggregation of expanded huntingtin in the brains of patients with Huntington disease. Prion 1, 26-31.

Choy J.S., Zhang Z.D., Pitsillides K., Sosa M. & Kassab G.S. (2014) Longitudinal hemodynamic measurements in swine heart failure using a fully implantable telemetry system. PLoS One 9, e103331.

Kiriazis H., Jennings N.L., Davern P., Lambert G., Su Y., Pang T., Du X., La Greca L., Head G.A., Hannan A.J. & Du X.J. (2012) Neurocardiac dysregulation and neurogenic arrhythmias in a transgenic mouse model of Huntington's disease. J Physiol 590, 5845-60.

Laferriere F., Tixador P., Moudjou M., Chapuis J., Sibille P., Herzog L., Reine F., Jaumain E., Laude H., Rezaei H. & Beringue V. (2013) Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics. PLoS Pathog 9, e1003702.

Lajoie P. & Snapp E.L. (2010) Formation and toxicity of soluble polyglutamine oligomers in living cells. PLoS One 5, e15245.

Miller J., Arrasate M., Brooks E., Libeu C.P., Legleiter J., Hatters D., Curtis J., Cheung K., Krishnan P., Mitra S., Widjaja K., Shaby B.A., Lotz G.P., Newhouse Y., Mitchell E.J., Osmand A., Gray M., Thulasiramin V., Saudou F., Segal M., Yang X.W., Masliah E., Thompson L.M., Muchowski P.J., Weisgraber K.H. & Finkbeiner S. (2011) Identifying polyglutamine protein species in situ that best predict neurodegeneration. Nat Chem Biol7, 925-34.

Moller T. (2010) Neuroinflammation in Huntington's disease. J Neural Transm (Vienna) 117,1001-8.

Morton A.J., Rudiger S.R., Wood N.I., Sawiak S.J., Brown G.C., McLaughlan C.J., Kuchel T.R., Snell R.G., Faull R.L. & Bawden C.S. (2014) Early and progressive circadian abnormalities in Huntington's disease sheep are unmasked by social environment. Hum Mol Genet 23, 3375-83.

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Pokorny M, Juhas S, Juhasova J, Klima J, Motlik J, Klempir J, Havlik J. (2015) Telemetry physical activity monitoring in minipig´s model of Huntington´s disease. Cesk Slov Neurol N 78/111, 2S39-2S42.

Sapp E., Kegel K.B., Aronin N., Hashikawa T., Uchiyama Y., Tohyama K., Bhide P.G., Vonsattel J.P. & DiFiglia M. (2001) Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol 60, 161-72.

Scherzinger E., Lurz R., Turmaine M., Mangiarini L., Hollenbach B., Hasenbank R., Bates G.P., Davies S.W., Lehrach H. & Wanker E.E. (1997) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90, 549-58.

Tai Y.F., Pavese N., Gerhard A., Tabrizi S.J., Barker R.A., Brooks D.J. & Piccini P. (2007) Microglial activation in presymptomatic Huntington's disease gene carriers. Brain 130,1759-66.

Trager U., Andre R., Magnusson-Lind A., Miller J.R., Connolly C., Weiss A., Grueninger S., Silajdzic E., Smith D.L., Leavitt B.R., Bates G.P., Bjorkqvist M. & Tabrizi S.J. (2015) Characterisation of immune cell function in fragment and full-length Huntington's disease mouse models. Neurobiol Dis 73, 388-98.

Valekova I., Jarkovska K., Kotrcova E., Bucci J., Ellederova Z., Juhas S., Motlik J., Gadher J.S.& Kovarova H. (2016) Revelation of the IFNĮ, IL-10, IL-8 and IL-1ȕ as promisingbiomarkers reflecting immuno-pathological mechanisms in porcine Huntington's disease model. Journal of Neuroimmunology 293, 71-81.

Wild E., Magnusson A., Lahiri N., Krus U., Orth M., Tabrizi S.J. & Bjorkqvist M. (2011) Abnormal peripheral chemokine profile in Huntington's disease. PLoS Curr 3, RRN1231.

Willens S., Cox D.M., Braue E.H., Myers T.M. & Wegner M.D. (2014) Novel technique for retroperitoneal implantation of telemetry transmitters for physiologic monitoring in Gottingen minipigs (Sus scrofa domesticus). Comp Med 64, 464-70.

SI

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Charts and Figures

A) Minipigs brain (36m) B) Mouse brain (12w)Tg K104

Caudate nucleusR6/2 WT

Aggregates

Figure 1. Immunostaining with mouse monoclonal MW8 antibody. Immunohistochemical staining of aggregates in minipig brain coronal sections (A) and in mouse brain coronal sections (B). MW8 antibody was used.

Figure 2. Detection of polymer formation by filter retardation assay. 36 months old (K100,K48,K104,K103), 24 months old (K55,K56) minipig's cortex, and 5 weeks old R6/2 mice brains WT and TgHD were compared using EPR5526 anti N-terminal Htt antibody.

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Figure 3. The presence of oligomeric structures in cortex of 24 and 36 months old TgHDminipigs. A, Oligomeric structures in TgHDminipigs cortex samples detected by EPR5526 anti-N-terminal Htt antibody by smears in higher molecular weights and comparison with R6/2 mice brain samples. B, Cortex of 36 months old minipigs reveals no smears after using (H-300) C-terminal Htt antibody.

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Figure 4. Detection of oligomeric structures in TgHDminipigs compared to WT in additional tissues. Anti-N-terminal Htt antibody EPR5526 shows oligomeric structures by smear in higher molecular weights also in testes of TgHDminipigs, but not in heart or muscles.

Figure 5. Fragmented mtHtt detected in 24 and 36 months old TgHDminipigs compared to their WT siblings in different tissues by polyQ 3B5H10 antibody. Western blot analysis shows fragmented forms of mtHtt in A, cortex B, testes of TgHDminipigs. C, Comparing to muscles of TgHDminipigs, testes show increased amount of fragmented mtHtt of 24 as well as36 months old minipigs.

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Opt

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Opt

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Figure 6. Immunostainingwithmousemonoclonalantibody anti-MMP9. Immunohistochemical staining in minipig brain coronal sections and results of Tukey´s tests comparing the expression of MMP9 in the brain areas (caudate nucleus, putamen, somatosensory cortex and motor cortex) between wild type (WT) and transgenic (Tg) minipigs. Western blot of minipig cerebral cortex confirmed the observation from staining.

WT K103 TgHD K104

Caudate nucleus Caudate nucleus

Caudate nucleus - DARPP32100

WT 100

Putamen - DARPP32

WT100

Caudate nucleus - DARPP32WT

Putamen - DARPP32100 WT

90 Tg 90Blank

80 80

Tg 90Blank

80

90 **

80

70 70 70 70

60 60 60 60

50 50 50 50

40 40 40 40

30 30 30 30

20 20 20 20

10 10 10 10

0Minipigs (36m)

0Minipigs (36m)

0K48 K100 K64 K63 K103K104

Minipigs (36m)

0K48 K100 K64 K63 K103K104

Minipigs (36m)

Figure 7. Immunostaining with rabbit monoclonal antibody anti DARPP32. Immunohistochemical staining in minipig and results of Tukey´s tests comparing the expression of DARPP32 in the caudate nucleus and putamen between wild type (WT) and transgenic (Tg) minipigs.

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Figure 8. Cytokine profiling in secretome of microglial cells A-B) Level of IFNĮ in response to two different stimulation doses (LPS = 10 and 100 ng/ml) as well as two time points was significantly reduced in TgHD minipigs at the age of 36 months. C) Level of IL-10 was significantly lower in TgHD animals after stimulation for 24h (LPS = 100 ng/ml) at the age of 36 months. D) IL-8 level was significantly increased in non-stimulated microglial cells isolated from TG minipigs and their WT counterparts. P values indicate the level of significance.

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Figure 9. Cytokines profiling in cerebrospinal fluid A) Highly significant reduction of IFNĮwas observed in CSF of TgHD animals. Such changes were detectable from a very early time interval (from 9 months of minipig age and lasted at least up to 36 months of age). These time intervals correspond to early pre-symptomatic stages of HD. B) The level of IL-8 showed dynamic alterations in TgHD animals but not in WT. C) Level of IL-10 was significantly reduced in TgHD animals at the age of 36 months. P values indicate the level of significance.

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Figure 10. Peripheral IL-8 level in transgenic porcine HD model Elevated levels of IL-8 were detected in peripheral blood serum of TgHD minipigs at the age of 36 months.

Figure. 11. Behavioral tests of F0, F1 and F2 generations. The pigs were evaluated in each discipline using points, where 0 was the worst and 5 was the best.

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TgHD WT

Figure 12. TgHD and WT F1 generation minipig on a balance beam

.

Figure 13. Balance beam test of F0 and F1. The pigs were tested seven times within one month using points, where 5 meant crossing the beam, 4 –crossing it to the middle, 3-eating one biscuit from the beam, 2-put forelegs and rear-legs on the beam, 1-put just forelegs on the beam, 0-did not enter at all the beam.

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Figure 14. Balance beam test of F2 generation. The pigs were tested five days in a row, where5 meant crossing the beam, 4 –crossing it to the middle, 3-eating one biscuit from the beam, 2- put forelegs and rear-legs on the beam, 1-put just forelegs on the beam, 0-did not enter at all the beam.

Figure 15. The positioning of the collar for activity measurement in TgHD boar.


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Figure 16. Total acceleration of TgHD and WT animals between 4:40 and 5:30 a.m. during six following days. Each column (blue – WT, red - TgHD) represents averaged total acceleration of 5 animals (TgHD or WT).

26

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Exhibit 1 to Res e a r c h Ag r eem e nt

(Form of Material Transfer Agreement)


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MATERIAL TRANSFER AGREEMENT

MATERIAL TRANSFER AGREEMENT (this "Agre e m e n t "), dated as of [ ], by and between [ ] (the "PR O VIDER "), [ ] (the "PR O VIDER SCI E NTIST "), [ ], a [ ] (the "RECIPI E NT "), and [ ] (the "RECIPI E N T SCIENTIS T "). The address and other contact information of each party hereto is as set forth in Appendix A.

The parties hereto desire to set forth certain terms and conditions to govern the transfer of certain materials between the parties hereto.

In consideration of the mutual representations, warranties and covenants contained herein and other good and valuable consideration, the receipt and sufficiency of which are hereby acknowledged, the parties hereby agree as follows:

1. Def i n i t i o ns . For the purposes of this Agreement, capitalized terms used herein but not otherwise defined shall have the meanings set forth below:

(a) "HD R ESE A RC H A ND DE V E L O PM ENT " means any activity useful for the creation, development, manufacture or distribution of a product or service for the diagnosis, treatment, cure or prevention of Huntington's disease other than (i) the manufacture or distribution of any such product or service for sale or (ii) the sale of any such product or service. For the avoidance of doubt, HD Research and Development shall not include any right to (A) manufacture or distribute any such product or service for sale or (B) sell any such product or service (including any transfer of any such services or products made using intellectual property rights, whether or not for consideration, other than a transfer of any such services or products solely for research and development purposes without fee or profit).

(b) "M A TE R IA L " means ORIGINAL MATERIAL, PROGENY, and UNMODIFIED DERIVATIVES. The MATERIAL shall not include: (i) MODIFICATIONS or (ii) other substances created by the RECIPIENT through the use of the MATERIAL which are not MODIFICATIONS, PROGENY, or UNMODIFIED DERIVATIVES.

(c) "MOD I FI C AT I ON S " means substances created by the RECIPIENT which contain/incorporate the MATERIAL.

(d) "O R IGINAL M A T E RIAL " means [ ] [INSERT DESCRIPTION OF ORIGINAL MATERIAL].

(e) "PROGENY" means unmodified descendant from the MATERIAL, such as virus from virus, cell from cell, or organism from organism.

(f) "PROVIDER" has the meaning set forth in the preamble and is the organization providing the ORIGINAL MATERIAL.

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(g) "RECIPIEN T " has the meaning set forth in the preamble and is the organization receiving the ORIGINAL MATERIAL.

(h) "U N M O D I F IED D E R I V A TI V ES " means substances created by the RECIPIENTwhich constitute an unmodified functional subunit.

2. P r ov i s i o n of M at e r i a l ; Ow n ersh i p .

(a) P r ov i s i o n of M at e r i a l . Within a reasonable period of time following the execution of this Agreement by the parties hereto, the PROVIDER shall provide to the RECIPIENT [the amount of the Original Materials as is specified on S c h e du l e A] [MODIFY AS APPROPRIATE].

(b) Ow n ership .

(i) The PROVIDER retains ownership of the MATERIAL, including anyMATERIAL contained or incorporated in MODIFICATIONS.

(ii) The RECIPIENT retains ownership of: (A) MODIFICATIONS (except that, the PROVIDER retains ownership rights to the MATERIAL included therein) and (B) those substances created through the use of the MATERIAL or MODIFICATIONS, but which are not PROGENY, UNMODIFIED DERIVATIVES or MODIFICATIONS (i.e., do not contain the ORIGINAL MATERIAL, PROGENY, UNMODIFIED DERIVATIVES). If (1) a MODIFICATION referred to in (A) above or(2) a substance referred to in (B) above results from the collaborativeefforts of the PROVIDER and the RECIPIENT, joint ownership may be negotiated.

3. No n-E xc lus ive L ice nse; U se of Mat er ia l.

(a) No n -E x c l us i ve L i ce ns e . The PROVIDER hereby grants to the RECIPIENT a non- exclusive, non-transferable, non-assignable, paid-up license throughout the world to (i) replicate the MATERIAL and (ii) use the MATERIAL for the sole purpose of conducting HD RESEARCH AND DEVELOPMENT.

(b) U s e of M ater i a l . The RECIPIENT and the RECIPIENT SCIENTIST agree that the MATERIAL:

(i) is to be used solely for HD RESEARCH AND DEVELOPMENTpurposes only;

(ii) will not be used in human subjects, in clinical trials, or for diagnostic purposes involving human subjects;

(iii) is to be used only at the RECIPIENT organization and only in theRECIPIENT SCIENTIST's laboratory under the direction of the

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RECIPIENT SCIENTIST or others working under his/her direct supervision; and

(iv) will not be transferred to anyone else within the RECIPIENT organization without the prior written consent of the PROVIDER.

4. R e q u e s ts f or M a ter i al f r o m Th i rd P a r t i es . The RECIPIENT and the RECIPIENT SCIENTIST agree to refer to the PROVIDER any request for the MATERIAL from anyone other than those persons working under the RECIPIENT SCIENTIST's direct supervision.

5. D ist r ibut io n o f Su bst ances a nd Mod ific at io ns.

(a) The RECIPIENT and/or the RECIPIENT SCIENTIST shall have the right, without restriction, to distribute substances created by the RECIPIENT through the use of the ORIGINAL MATERIAL only if those substances are not PROGENY, UNMODIFIED DERIVATIVES or MODIFICATIONS.

(b) Under an agreement at least as protective of the PROVIDER's rights, the RECIPIENT may distribute MODIFICATIONS to third parties for HD RESEARCH AND DEVELOPMENT purposes only.

(c) Without written consent from the PROVIDER, the RECIPIENT and/or the RECIPIENT SCIENTIST may not distribute MODIFICATIONS to third parties for any purpose other than HD RESEARCH AND DEVELOPMENT. It is recognized by the RECIPIENT that any use of MODIFICATIONS for any purposes other than for HD RESEARCH AND DEVELOPMENT may require a commercial license from the PROVIDER and the PROVIDER has no obligation to grant a commercial license to its ownership interest in the MATERIAL incorporated in the MODIFICATIONS. Nothing in this paragraph, however, shall prevent the RECIPIENT from granting commercial licenses under the RECIPIENT's intellectual property rights claiming such MODIFICATIONS, or methods of their manufacture or their use.

6. T h e R ec i p i e n t ' s A ck n o w l e d ge m e n t o f I n t e ll e c tual P r ope r ty R i g h ts . The RECIPIENT acknowledges that the MATERIAL is or may be the subject of a patent application. Except as provided in this Agreement, no express or implied licenses or other rights are provided to the RECIPIENT under any patents, patent applications, trade secrets or other proprietary rights of the PROVIDER, including any altered forms of the MATERIAL made by the PROVIDER. In particular, no express or implied licenses or other rights are provided to use the MATERIAL, MODIFICATIONS, or any related patents of the PROVIDER for any purpose other than HD RESEARCH AND DEVELOPMENT.

7. R e q u i re m e n t to N e g ot i ate C o m m e r c i al L i ce ns e t o Us e t h e M at e r i a l s . If the RECIPIENT desires to use or license the MATERIAL or MODIFICATIONS for any purpose other than HD RESEARCH AND DEVELOPMENT, the RECIPIENT agrees,

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in advance of such use, to negotiate in good faith with the PROVIDER to establish the terms of a

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commercial license. It is understood by the RECIPIENT that the PROVIDER shall have no obligation to grant such a license to the RECIPIENT, and may grant exclusive or non- exclusive commercial licenses to others, or sell or assign all or part of the rights in the MATERIAL to any third party(ies), subject to any pre-existing rights held by others and obligations to the Federal Government.

8. R i g h t o f t h e R ec i p i e n t t o F il e P at e n t A pp li ca t i o ns . The RECIPIENT is free to file patent application(s) claiming inventions made by the RECIPIENT through the use of the MATERIAL but agrees not to file any patent application claiming, the MATERIAL, MODIFICATIONS or method(s) of manufacture of the MATERIAL.

9. No W arrant i e s . Any MATERIAL delivered pursuant to this Agreement is understood to be experimental in nature and may have hazardous properties. The PROVIDER MAKES NO REPRESENTATIONS AND EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED. THERE ARE NO EXPRESS OR IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR THAT THE USE OF THE MATERIAL WILL NOT INFRINGE ANY PATENT, COPYRIGHT, TRADEMARK, OR OTHER PROPRIETARY RIGHTS.

10. As s u m p t i o n o f Liabil i t y ; Ind e m nific a t i o n . Except to the extent prohibited by law, the RECIPIENT assumes all liability for damages which may arise from its use, storage or disposal of the MATERIAL. The PROVIDER will not be liable to the RECIPIENT for any loss, claim or demand made by the RECIPIENT, or made against the RECIPIENT by any other party, due to or arising from the use of the MATERIAL by the RECIPIENT, except, to the extent permitted by law, when caused by the gross negligence or willful misconduct of the PROVIDER. Except to the extent prohibited by law, the RECIPIENT will indemnify the PROVIDER (and its officers, faculty, trustees, and agents) against any loss, claim or demand suffered by the PROVIDER due to or arising from the use of the MATERIAL by the RECIPIENT, except, to the extent permitted by law, when caused by the gross negligence or willful misconduct of the PROVIDER.

11. No Ef f ect on Pub li c a t i o n; A c k n o w l edgement o f Source o f t h e M a t er i a l . This Agreement shall not be interpreted to prevent or delay publication of research findings resulting from the use of the MATERIAL or the MODIFICATIONS. The RECIPIENT SCIENTIST agrees to provide appropriate acknowledgement of the source of the MATERIAL in all publications.

12. C o m pl i a n ce w i th L a w . The RECIPIENT agrees to use the MATERIAL in compliance with all applicable statutes and regulations, including Public Health Service and National Institutes of Health regulations and guidelines such as, for example, those relating to research involving the use of animals or recombinant DNA.

13. Te r mi na t i o n . This Agreement will terminate upon a material breach of any representation, warranty or covenant of this Agreement by the RECIPIENT and such breach is not remedied within 45 days of the receipt by the RECIPIENT of notice of

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such breach from the PROVIDER. Upon the termination of this Agreement, the RECIPIENT will (a) discontinue its use of the MATERIAL and will, upon direction of the

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PROVIDER, return or destroy any remaining MATERIAL and (b) at the RECIPIENT's discretion, either destroy the MODIFICATIONS or remain bound by the terms of this Agreement as they apply to MODIFICATIONS.

14. S u rv i val o f C e r ta i n Prov i s i o n s . S e ct i on 2( b ) S e ct i on 6 , S e c t i on 9 , S e c t i on 1 0 , S e c t i on 1 6 , Sec t i o n 17 , Sec t i o n 19 , Sect i o n 20 , Sect i o n 21 and Sec t i o n 22 shall survive any termination of this Agreement.

15. C o st to Pr o v i de Ma t e r i a l . The MATERIAL is provided at no cost.]/[The Material is provided subject to a transmittal fee in the amount of $[ ] which amount solely covers the PROVIDER's preparation and distribution costs.] [SELECT AS APPROPRIATE]

16. No t i ces . Any notice required or permitted to be given by this Agreement shall be in writing and shall be delivered by personal delivery, facsimile (provided the sender has evidence of successful transmission) or next day courier service to the party at its address set forth in A p p e n d i x A or such other address as the party may, by notice, specify. Any notice so delivered shall be deemed to be given, delivered and received, if delivered by personal delivery, on the day of delivery and if delivered by facsimile or courier service, on the day following dispatch.

17. A s s i g n m e n t . Neither the Recipient nor the Recipient Scientist may assign this Agreement without the written consent of the Provider.

18. I nco r p o r a t i o n o f A pp e n d i c e s a nd E xh i b i t s; E nt i r e A g r e e m e n t ; A m e n d m e n t . The appendices and exhibits identified in this Agreement are incorporated herein by reference and made a part hereof. If anything in any appendix or exhibit attached to this Agreement conflicts with any terms or conditions set forth in the body of this Agreement, the terms and conditions set forth in the body of this Agreement shall control. This Agreement constitutes the entire agreement among the parties hereto relating to the subject matter hereof and all prior understandings and agreements relating to the subject matter hereof are superseded hereby. This Agreement may not be amended except by a document signed by each of the parties hereto.

19. No W a i v e r . Any failure of a party hereto to enforce any provision of this Agreement shall not be deemed a waiver of its right to enforce such provision on any subsequent occasion. No waiver of any provision of this Agreement shall be valid unless it is in writing and is executed by the party against whom such waiver is sought to be enforced. A waiver by any of the parties hereto of any provision of this Agreement will not be construed to be a waiver of any succeeding breach thereof or of any other provision of this Agreement.



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21. Interp r e t a t i o n; Heading s . The word "i n c l u d i ng " shall mean "includi n g w i t h o ut l i m i tat i on ". All pronouns and any variations thereof refer to the masculine, feminine or neuter, singular or plural, as the context may require. All terms defined in this Agreement in their singular or plural forms have correlative meanings when used herein in theirplural or singular forms, respectively. Headings used in this Agreement are for convenience of reference only and are not intended to influence the interpretation hereof.

22. N o S t r i c t Co nst r u c t i o n . The parties hereto have participated jointly in the negotiation and drafting of this Agreement. In the event of an ambiguity or question of intent or interpretation arises, this Agreement shall be construed as if drafted jointly by the parties hereto, and no presumption or burden of proof shall arise favoring or disfavoring any of the parties hereto by virtue of the authorship of any of the provisions of this Agreement.


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In witness to the foregoing, the Parties have executed this Material Transfer Agreement as of the date first written above.

PROVIDER:

[ ]

By: Name:Title:

RECIPIENT:

[ ]

By: Name: Title:

PROVIDER SCIENTIST:

[ ]

RECIPIENT SCIENTIST:

[ ]


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Ap p e n d ix A to M ater i al T ransfer Ag r e e me n t

(Original Materials)

Provider

Name: Telephone:

ContactPerson/Title: Fax:

Address: E-Mail:

Provider Scientist

Name: Telephone:


Address: E-Mail:

Recipient

Name: Telephone:


Address: E-Mail:

Recipient Scientist

Name: Telephone:


Address: E-Mail:

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Exhibit 2 to Res e a r c h Ag r eem e nt

(Form of Material Transfer Agreement for Research Materials)


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MATERIAL TRANSFER AGREEMENT

MATERIAL TRANSFER AGREEMENT (this "Agre e m e n t "), dated as of [ ] (the "E f f ect i ve D a te "), by and between CHDI Foundation, Inc., a New Jersey corporation (the "F o u n da t i o n "), and [ ], a [ ] corporation (the "Pr o v i d er ").

The Foundation supports basic, applied and clinical research aimed at finding diagnoses, treatments and cures of Huntington's disease ("H D ") and has access to a variety of relevant research tools including in vitro and in vivo assays and animal models.

The Provider possesses certain materials and is willing to supply the Foundation with such materials to enable the Foundation to perform, or have performed, research and development related to HD.

The parties hereto desire to set forth certain terms and conditions to govern the transfer of certain materials from the Provider to the Foundation and the use of such materials by the Foundation.

In consideration of the mutual representations, warranties and covenants contained herein and other good and valuable consideration, the receipt and sufficiency of which are hereby acknowledged, the parties hereto hereby agree as follows:

1. Def i n i t i o ns . For the purposes of this Agreement, capitalized terms used herein but not otherwise defined shall have the meanings set forth below:

(a) "F o u n da t i o n C o ll a b o rators " means those third parties to whom the Foundation grants the right to use the Material for HD Research and Development, including any entity collaborating with the Foundation in the conduct of HD Research and Development and/or fee for service laboratories providing services to the Foundation in the furtherance of the Foundation's conduct of HD Research and Development.

(b) "HD R e s e a rch a n d D e v e l o p m e n t " means any activity useful for the creation, development, manufacture or distribution of a product or service for the diagnosis, treatment, cure or prevention of HD other than (i) the manufacture or distribution of any such product or service for sale or (ii) the sale of any such product or service. For the avoidance of doubt, HD Research and Development shall not include any right to (A) manufacture or distribute any such product or service for sale or (B) sell any such product or service.

(c) "M at e r i a l " means the Original Materials, Progeny and Unmodified Derivatives.The Material shall not include: (i) Modifications or (ii) other substances created by the Foundation or a Foundation Collaborator through the use of the Material which are not Modifications, Progeny or Unmodified Derivatives.

(d) "Mo d i f i c a t i o ns " means substances created by the Foundation or a Foundation


Execution Collaborator which contain/incorporate the Material.

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(e) "Or ig inal Mater ia ls" means the materials described on Schedu le A.

(f) "Pr o gen y " means unmodified descendant from the Material, such as virus from virus, cell from cell, or organism from organism.

(g) "U n m od i fied De r i va t i v e s " means substances created by the Foundation or a Foundation Collaborator which constitute an unmodified functional subunit or product expressed by the Original Materials. Some examples include: subclonesof unmodified cell lines, purified or fractionated subsets of the Original Materials, proteins expressed by DNA/RNA supplied by the Provider, or monoclonal antibodies secreted by a hybridoma cell line.

2. P rov is io n of t he Or ig inal Mat er ia ls; No Wa rra nt ies; Ow ne rsh ip .

(a) P r ov i s i o n of t h e O r i g i nal M a ter i a l s; No W a r ra n t i es .

(i) P r ov i s i o n of t h e O r i g i nal Mate r i a l s . Within a reasonable period of time following the execution of this Agreement by the parties hereto, the Provider shall provide to the Foundation (or, as designated by the Foundation in writing, to a Foundation Collaborator) [the amount of the Original Materials as is specified on Schedule A] [MODIFY AS APPROPRIATE]. [The Foundation shall reimburse the Provider for the cost of the delivery of the Original Materials to the Foundation (or, as designated by the Foundation in writing, to a Foundation Collaborator).]/[The Original Material is provided at no cost.]/[The Original Material is provided subject to the payment of a transmittal fee by the Foundation in the amount of $[ ] which is the Provider's reasonable direct costs associated with so providing the Original Material.] [SELECT AS APPROPRIATE]

(ii) No W arrant i e s . Any Original Materials provided to the Foundation hereunder are understood to be experimental in nature and may have hazardous properties. THE ORIGINAL MATERIALS ARE PROVIDED "AS-IS" AND THE PROVIDER MAKES NO REPRESENTATIONS AND EXTENDS NO WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED. THERE ARE NO EXPRESS OR IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, OR THAT THE USE OF THE MATERIAL WILL NOT INFRINGE ANY PATENT, COPYRIGHT, TRADEMARK, TRADE SECRET OR OTHER PROPRIETARY RIGHT.

(b) Ownership.

(i) O w n e r sh i p o f t h e M at e r i a l . As between the Provider and the Foundation or any Foundation Collaborator, the Provider shall retain ownership of the

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Material, including any Material contained or incorporated in any Modification.

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(ii) O w n e r sh i p o f M od i f i c a t i o n s a n d O t h er S u b s tan c es . As between the Provider and the Foundation or any Foundation Collaborator, the Foundation or Foundation Collaborator, as the case may be, retains ownership of: (A) Modifications (except that the Provider retains ownership rights to the Material included therein) and (B) those substances created through the use of the Material or Modifications, but which are not Modifications, Progeny or Unmodified Derivatives (i.e., do not contain the Original Materials, Progeny or Unmodified Derivatives).

3. No n -E x c l us i ve L i ce ns e; Stor a ge of t h e M at e r i al and C o n d u ct of t h e H D R e s e a rch a n d D e v e l o p m e n t at F ou n d a t i on C o ll a b orat o r s ; U s e of t h e M at e r i a l .

(a) No n -E x c l us i ve L i ce ns e . The Provider hereby grants to the Foundation a non- exclusive, non-transferable, non-assignable, paid-up license throughout the world to (i) replicate the Material and (ii) use the Material for the sole purpose of conducting HD Research and Development.

(b) St o ra g e o f t h e Ma t er i a l and C o n duct o f t h e HD Resear c h a n d D e v e l op ment at F o u n da t i o n Col l a b o rat o rs . The Provider (i) acknowledges that the Foundationdoes not have any (A) material storage, handling or distribution capabilities or (B)laboratory capabilities and (ii) acknowledges and agrees that (A) the Materialshall be stored, handled and distributed on behalf of the Foundation by a Foundation Collaborators engaged by the Foundation to store, handle and distribute the Material, (B) the programs of HD Research and Development shall be conducted by one or more Foundation Collaborators and (C) the Material may be transferred, and the rights granted to the Foundation pursuant to Se c t i o n 3(a) of this Agreement may be sublicensed, to the Foundation Collaborators.

(c) Use o f t he Mat eria l. The Foundation hereby agrees:

(i) to use the Material for the sole purpose of conducting HD Research andDevelopment and for no other purpose;

(ii) to use the Material in compliance with all applicable laws, rules and regulations;

(iii) not to use the Material in human subjects, in clinical trials or for diagnostic purposes involving human subjects;

(iv) except as expressly permitted by this Agreement, not to transfer theMaterial or the Information to any third party; and

(v) cause each Foundation Collaborator to agree to comply with each of S e ct i on 3( c )( i ) , S e ct i on 3( c ) ( i i ) , S e ct i on 3( c ) ( iii ) and S e ct i on 3( c )( iv ) of this Agreement.

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4. Int e ll ectu a l Pr o pe r t y ; Acknowledgements of the Foundation in Respect of IntellectualProperty.

(a) Intellectual Property. The Provider hereby acknowledges and agrees that nothing in this Agreement gives the Provider any ownership interests or intellectual property or other rights in any (i) any substances created by the Foundation or any Foundation Collaborator through the use of the Material other than as expressly provided in S e c t i on 2( b )(i i ) of this Agreement or (ii) any results, discoveries, inventions, formulations, know-how, methods, technological developments, enhancements, modifications, improvements, works of authorship, data or collections of data conceived, discovered, invented, made or first reduced to practice by the Foundation or any Foundation Collaborator through the use of the Material. The Provider hereby further acknowledges and agrees that the Foundation and Foundation Collaborators are free to file patent application(s) claiming inventions conceived, discovered, invented, made or first reduced to practice by the Foundation or any Foundation Collaborator through the use of the Material; provided, that, the Foundation agrees, and shall cause each Foundation Collaborator to agree, not to file any patent application containing a composition of matter claim for the Material, per se.

(b) A ck n o w l e d g e m e n ts of t h e F ou n d a t i on i n R es p e c t o f I n t e ll e c tual Prop er t y . The Foundation acknowledges, and shall cause each Foundation Collaborator to acknowledge, that the Material is, or may be, the subject of a patent application. Except as expressly provided in this Agreement, the Foundation hereby acknowledges and agrees, and shall cause each Foundation Collaborator to acknowledge and agree, that no express or implied licenses or other rights are provided to the Foundation or any Foundation Collaborator under any patents, patent applications, trade secrets or other proprietary rights of the Provider, including any altered forms of the Material made by the Provider. In particular,the Foundation hereby acknowledges and agrees, and shall cause each FoundationCollaborator to acknowledge and agree, that no express or implied licenses or other rights are provided to use the Material, Modifications or any related patents of the Provider for any purpose other than HD Research and Development.

5. A ck n o w l e d g e m e n t of t h e S o u r c e of t h e M at e r i a l . The Foundation agrees, and shall cause each Foundation Collaborator to acknowledge and agree, to provide appropriate acknowledgement of the source of the Material in all publications related to HD Research and Development conducted using the Material.

6. A s s u m p t i o n o f L i a bil i t y; I n de m ni f ic a t i o n; L im i t at i o n o n D a m ag e s .

(a) As s u m p t i o n o f Liabil i t y ; Ind e m nific a t i o n . Except to the extent prohibited by law, the Foundation assumes all liability for damages to the extent due to or arising from the use, storage or disposal of the Material by the Foundation or a Foundation Collaborator. The Provider will not be liable to the Foundation for

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any loss, claim or demand made by the Foundation or a Foundation Collaborator,

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or made against the Foundation or a Foundation Collaborator by any other party, to the extent due to or arising from the use, storage or disposal of the Material by the Foundation or a Foundation Collaborator. Except to the extent prohibited by law, the Foundation will defend and indemnify the Provider (and its directors, officers, employees, trustees, shareholders, members and agents) against any loss, claim or demand (including attorneys' fees and cost of defense and the enforcement of this provision) suffered by the Provider to the extent due to or arising from the use, storage or disposal of the Material by the Foundation or a Foundation Collaborator.

(b) Lim i t a t i o n o n D a m a g es . NOTWITHSTANDING ANY OTHER PROVISION OF THIS AGREEMENT, NEITHER PARTY NOR ITS AFFILIATES WILL BE LIABLE TO THE OTHER PARTY OR ITS AFFILIATES FOR ANY CONSEQUENTIAL, SPECIAL, INDIRECT, INCIDENTAL, PUNITIVE OR EXEMPLARY DAMAGES OR OTHER SIMILAR OR LIKE DAMAGES (INCLUDING LOSS OF PROFITS) UNDER THIS AGREEMENT EVEN IF SUCH PARTY OR AFFILIATE HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES; PROVIDED, THAT, NOTHING IN THIS AGREEMENT SHALL EXCLUDE OR LIMIT THE LIABILITY OF EITHER PARTY FOR (I) DEATH OR PERSONAL INJURY OR (II) FRAUD.

7. Terminat io n; Effect o f Terminat io n; Surviva l of Cer t ain Pro visio ns.

(a) T e r mi n at i o n . This Agreement will automatically terminate upon a material breach of any representation, warranty or covenant of this Agreement by the Foundation and such breach is not remedied within 45 days of the receipt by the Foundationof notice of such breach from the Provider.

(b) E ff e c t o f Te r mi n a t i o n . Upon any termination of this Agreement, the Foundation(i) will immediately discontinue its use of the Material and any Modifications and(ii) will immediately and appropriately destroy or discard any remaining Material and any Modifications.

(c) S u rv i val o f C e r ta i n Prov i s i o n s . This S e ct i on 7 and each of S e ct i on 1 , S e c t i on 2( b ) , S e ct i on 4 , S e c t i on 5 , S e c t i on 6 , S e c t i on 8 , S e c t i on 9 , S e c t i on 1 0 , S e ct i on 1 1 , Sec t i o n 12 , Sec t i o n 13 , Se c t i o n 14 and Sec t i o n 15 of this Agreement shall survive any termination of this Agreement.

8. No t i ces . Any notice required or permitted to be given by this Agreement shall be in writing and shall be delivered by personal delivery, facsimile (provided the sender has evidence of successful transmission) or next day courier service. Any notice so delivered shall be deemed to be given, delivered and received, if delivered by personal delivery, on the day of delivery and if delivered by facsimile or courier service, on the day following dispatch. All such notices are to be given or made to the parties at the following

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addresses (or to such other address as any party may designate by a notice given in accordance with the provisions of this section):

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If to the Foundation to:

CHDI Foundation, Inc.c/o CHDI Management, Inc.350 Seventh Avenue, Suite 200New York, NY 10001Attention: Ruth BasuFax: 212-239-2101

With a copy to:

CHDI Foundation, Inc.c/o CHDI Management, Inc.350 Seventh Avenue, Suite 200New York, NY 10001Fax: 212-239-2101Attention: General Counsel

If to the Provider to:

[ ][ ][ ]Attention: [ ]Fax: [ ]

9. A s s i g n m e n t . The Foundation may not assign this Agreement without the written consent of the Provider.

10. I n c o r p o r a t i o n o f A pp e n d i c e s, E x h i b i t s a nd S c h e d u l e s; E nt i r e A g r e e m e n t ; Amendment.The appendices, exhibits and schedules identified in this Agreement are incorporated herein by reference and made a part hereof. If anything in any appendix, exhibit or schedule attached to this Agreement conflicts with any terms or conditions set forth in the body of this Agreement, the terms and conditions set forth in the body of this Agreement shall control. This Agreement constitutes the entire agreement among the parties hereto relating to the subject matter hereof and all prior understandings and agreements relating to the subject matter hereof are superseded hereby. This Agreement may not be amended except by a document signed by each of the parties hereto.

11. No W a i v e r . Any failure of a party hereto to enforce any provision of this Agreement shall not be deemed a waiver of its right to enforce such provision on any subsequent occasion. No waiver of any provision of this Agreement shall be valid unless it is in writing and is executed by the party against whom such waiver is sought to be enforced. A waiver by any of the parties hereto of any provision of this Agreement will not be construed to be a waiver of any succeeding breach thereof or of any other provision of this Agreement.


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13. Interp r e t a t i o n; Heading s . The word "including" shall mean "including without limitation". All pronouns and any variations thereof refer to the masculine, feminine or neuter, singular or plural, as the context may require. All terms defined in this Agreement in their singular or plural forms have correlative meanings when used herein in theirplural or singular forms, respectively. Headings used in this Agreement are forconvenience of reference only and are not intended to influence the interpretation hereof.

14. N o S t r i c t Co nst r u c t i o n . The parties hereto have participated jointly in the negotiation and drafting of this Agreement. In the event of an ambiguity or question of intent or interpretation arises, this Agreement shall be construed as if drafted jointly by the parties hereto, and no presumption or burden of proof shall arise favoring or disfavoring any of the parties hereto by virtue of the authorship of any of the provisions of this Agreement.

15. G o v erning La w . This Agreement shall be governed by and construed in accordance with the domestic laws of the State of New York without giving effect to any choice or conflict of law provision or rule (whether of the State of New York or any otherjurisdiction) that would cause the application of the laws of any jurisdiction other than theState of New York.


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In witness to the foregoing, the parties hereto have executed this Material TransferAgreement as of the date first written above.

PROVIDER:

[_ _][INSERT NAME OF THE PROVIDER

By: Name:Title:

FOUNDATION:

CHDI Foundation, Inc.

By: Name:Title:


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Schedule A to M ateri a l T ra n sf e r Ag r eement

(Original Materials)

I tem De s c r iption of O r iginal Mate r i a l A m ount

1

2

3

4

5

6

7

[LIST/PROVIDE DESCRIPTION AND AMOUNT OF EACH MATERIAL TO BE PROVIDED]

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