ZFNs: Zinc finger chimeric endonucleases -- a way to make a site-specific double-stranded break at a

chosen site

• Link zinc fingers to a nonspecific endonuclease (a dimer).

• Use four fingers per endonuclease monomer, so effectively have a 24-basepair recognition site -- long enough to specify unique site in mammalian genomes.

• Zinc fingers that recognize specific triplets have been identified (most are in proprietary database of a biotech company called Sangamo BioSciences).

• Use for gene therapy:– Urnov et al. (2005) Nature 435: 646-651 (News and Views, pgs 577-579); Kandavelou et al. (2005) Nature Biotechnology 23: 686-687.

Zinc finger endonuclease-mediated gene targeting in human cells

Kandavelou et al. (2005) Nature Biotechnology 23: 686-687

• Zinc fingers that specifically target particular 3 basepair sequences were developed by phage display and other selection methods.

• Because they are modular recognition units, they can be linked together in any order to create a reagent that specifically recognizes any desired DNA sequence.

Each “N” is a subunit of a dimeric endonuclease.

Gene Therapy

• Treat genetic diseases by correcting gene defect

• Current gene therapy protocols involve gene addition to insert therapeutic genes randomly in genome using viral vectors -- random insertions can activate oncogenes as seen in gene therapy trials to treat X-SCID.

• Zinc finger endonuclease-mediated gene targeting uses cell’s homology-directed gene editing process rather than gene addition.

• Dimerized enzyme introduces double-stranded break near site of mutation. Homologous recombination machinery repairs damage using wild-type gene that was introduced along with zinc finger endonuclease.

Gene correction

High, 2005, Nature 435; 577-579

Life-long supply of anti-HIV

neutralizing antibodies

LymphocytesTarget HIV

Engineering Immunity against HIVDavid Baltimore, PI, Bill and Melinda Gates Foundation

Hematopoietic stem cells

(Lentivirus)

Clicker question

Is it necessary to isolate autologous (a person’s own) hematopoietic stem cells in the Engineering Immunity approach?

1) Yes2) No

Engineering immunity to Engineering immunity to HIVHIV

•Hematopoietic stem cell (HSC) transduction with a Hematopoietic stem cell (HSC) transduction with a lentiviral vector lentiviral vector (1)(1)

• Cell-specific expression Cell-specific expression (2)(2)

• May be used to express engineered anti-HIV May be used to express engineered anti-HIV moleculesmolecules

• Does not rely on host immune systemDoes not rely on host immune system

• Not limited to natural antibody architectureNot limited to natural antibody architecture

(1)(1) Yang, L., and D. Baltimore, PNAS, 2005. 102: 4518-23. Yang, L., and D. Baltimore, PNAS, 2005. 102: 4518-23. (2)(2) Lois, C., et al., Science, 2002. 295: 868-72.Lois, C., et al., Science, 2002. 295: 868-72.

scFv (single-chain Fv)

Intact IgG antibody

Smaller versions of antibodies against the CCR5 binding site on gp120 neutralize HIV more effectively than intact

antibodies, but the immune system only produces intact antibodies

These are idealized data from an in vitro neutralization assay.

Steric restrictions on antibody access to the coreceptor binding site on gp120

Burton et al. (2005) PNAS 102, 14943-8

scFv and Fab versions of anti-CCR5 antibodies can fit in space between gp120 and target cell membrane. Intact IgG is too big. Explains why scFv and Fabs of anti-CCR5 antibodies neutralize virus better than IgG versions. People don’t normally make scFv and Fab versions of antibodies, but they could be programmed to do so in an Engineering Immunity approach.

Antibody design

News feature, Nature (2007) volume 446, pp. 964-966

Alternative architectures for new anti-HIV moleculesAlternative architectures for new anti-HIV molecules

TraditionTraditional al

antibodyantibody

Anthony West, Josh Klein (former Bi1 TA)

•Single chain bivalent Gly-Ser linker is highly Single chain bivalent Gly-Ser linker is highly flexibleflexible

• May favor 1:1 over 2:1 stoichiometryMay favor 1:1 over 2:1 stoichiometry

•Diabodies exhibit restricted torsion angles Diabodies exhibit restricted torsion angles (1)(1)

• May favor 2:1 over 1:1 stoichiometryMay favor 2:1 over 1:1 stoichiometry

(1)(1) Lawrence, L.J., A.A. Kortt, et al., FEBS Lett, 1998. 425(3): 479-84.Lawrence, L.J., A.A. Kortt, et al., FEBS Lett, 1998. 425(3): 479-84.

Selection of combining sites may Selection of combining sites may lead to different stoichiometrieslead to different stoichiometries

Different types of bispecific reagentsDifferent types of bispecific reagents

We know very little about how HIV envelope spikes are We know very little about how HIV envelope spikes are distributed on virionsdistributed on virions

Zhu et al., 2003, PNAS 100: 15182-15187Zhu et al., 2003, PNAS 100: 15182-15187

Mutant SIV~75 trimers

HIV7-14 trimers

100 nm

Electron Electron tomography tomography

of HIV of HIV envelope envelope

glycoprotein glycoprotein trimers on trimers on

virionsvirions

Antibody binding sites on model of trimeric viral spike

Burton, Dennis R. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 14943-14948

2G12 Fabs are domain swapped to form a rigid Fab2 unit.

Calarese et al., 2003, Science 300: 2065-71.

2G12 also forms domain-swapped dimers (two IgGs; four Fabs)

West et al., 2009,

J. Virol. 83: 98-104

The dimeric form of 2G12 IgG is more effective in neutralizing HIV

Anthony West, Priyanthi Peiris, Joshua Klein

Dimer IC50: 0.94 ± 0.053 nM Monomer IC50: 43 ± 4.9 nM

Dimer IC50: 0.43 ± 0.066 nMMonomer IC50: 30 ± 3.2 nM

2G12 dimer neutralizes more effectively than 2G12 monomer

CAVD NAb Core, Beth Huey Tubman, Anthony West, Priyanthi Gnanapragasam

Env clone

Monomer

IC 50 (g/ml)

Dimer

IC 50 (g/ml)

Monomer IC50/

Dimer IC50

SC422661.8 2.8 <0.05 56TRO.11 0.58 <0.05 12PVO.4 2.6 <0.05 52QH0692.42 6.5 <0.05 1306535.3 20 0.35 57TRJO4551.58 >100 0.16 620WITO4160.33 6.1 <0.05 120

Average 6.2 0.08 79

Life-long supply of anti-HIV

neutralizing antibodies

LymphocytesTarget HIV

Engineering Immunity against HIVDavid Baltimore, PI, Bill and Melinda Gates Foundation

Hematopoietic stem cells

Is it practical to isolate hematopoietic stem cells from every person to be treated?

Baltimore/Yang/Wang Approach to Target Recombinant Lentiviruses to Hematopoietic Stem

Cells (HSCs)

binding induces endocytosis

endosomal compartme

ntlow pH

CD34

Anti-CD34 antibody targets lentivirus to HSCs.

Recombinant lentivirus with membrane-bound anti-CD34 plus flu hemagglutinin, which has been mutated so that it no longer binds sialic acids.

Receptor-mediated endocytosis brings recombinant lentivirus to an acid endosome.

Low pH triggers hemagglutinin to fuse with endosomal membrane.

Viral capsid released into cytoplasm of cell.

Potentially safer method than lentiviral or retroviral vectors: Use adeno-associated virus (AAV) to deliver genes

• AAV infects humans and primates– Doesn’t cause disease -- is a satellite virus

• ssDNA virus, stably integrates into host genome at specific site on chromosome 19 • Integrative capacity removed from AAV-based gene therapy vectors. Form episomal (not in a chromosome) concatamers in host cell nucleus

– Concatamers retained in non-dividing cells– Concatamers lost during cell division in non-dividing cells

T = 1 icosahedral virus

Crystal structure described inXie et al., 2002, PNAS 99 10405-10

Long term supply of

anti-HIV neutralizing

antibodies

Engineering Immunity against HIV

Gene for anti-HIV NAb or Ab-like protein

Engineered recombinant

Adeno-Associated Virus

Make improved antibody/antibody-like protein reagents starting with known broadly neutralizing anti-HIV antibodies.

Persistent muscle cell expression

Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against

SIV infection in monkeysJohnson et al., 2009, Nature Medicine published online 17 May 2009;

doi:10.1038/nm.1967

• Immunized rhesus macaques with AAV vectors carrying neutralizing antibody constructs• Intramuscular injections

– achieved µg/ml concentrations in the blood– single injection resulted in long-term (>1 year) expression of biologically-active antibodies

• Four weeks after intramuscular injection, injected SIV • After six more weeks

– 6/6 control monkeys infected after challenge. 4/6 developed AIDS after ~one year – 6/9 AAV-injected monkeys were protected against infection, none developed AIDS after ~one year

Extra credit opportunitySee course website for details

• Submit an idea for how to prevent or cure HIV infection (due Friday 6/10/11).

• If you wish to use an “Engineering Immunity” gene therapy approach (as discussed this lecture), follow the instructions on the Bi1 assignments page of the course website for describing your idea. • If you have a different sort of idea, follow the guidelines described for a Grand Challenges Exploration grant: go to http://www.gcgh.org/Explorations/Pages/ApplicationInstructions.aspx and and click on "Read Round 7 topics."

– The Bill and Melinda Gates Foundation is inviting anyone and everyone to submit short (maximum of two pages) proposals describing new ideas that are “off the beaten track” and “daring in premise.” – Applicants chosen for funding will receive $100,000 to carry out the research. Round 7 submissions were due May 19, 2011, but there will be another round of submissions in Fall 2011. – If anyone is interested in submitting his/her idea for Round 8, we will be happy to discuss it further with you and/or help you write the actual proposal. – If you are chosen for funding, we will find a laboratory at Caltech where you can conduct your research. If you are not chosen for funding, but are committed to trying your idea, we will help you find a way to test your idea in a lab at Caltech or elsewhere as a SURF project.

Extra slides

3D reconstruction of SIV virions by cryo-electron tomography Zanetti, G., et al., PLoS Pathogens 2, e83 (2006)

Trimeric HIV envelope spike structures have been examined on viruses using cryoelectron tomography

The HIV envelope spike is a trimer of gp120/gp41* heterodimers.*gp (glycoprotein) 120 is 120 kDa; gp41 is 41 kDa.

Zanetti, G., et al., PLoS Pathogens 2, e83 (2006) Zhu, P. , et al., Nature 441, 847 (2006)

The resulting structure is low resolution, but shows that the trimer should be accessible to antibodies

Clicker question: Companies have spent a lot of money trying to develop a soluble form of CD4 that could be injected into an HIV-positive patient. The reasoning behind this approach is that…(a) Soluble CD4 would compete with membrane bound CD4 to prevent gp120 from binding T cells. (b) Injected soluble CD4 is unlikely to trigger an immune reaction.(c) gp120 can’t mutate its CD4 binding site without reducing its ability to infect T cells. (d) All of the above. (e) None of the above.

CCR5

gp120

CD4

Clicker question: Companies have spent a lot of money trying to develop a soluble form of CD4 that could be injected into an HIV-positive patient. The reasoning behind this approach is that…(a) Soluble CD4 would compete with membrane bound CD4 to prevent gp120 from binding T cells. (b) Injected soluble CD4 is unlikely to trigger an immune reaction against it.(c) gp120 can’t mutate its CD4 binding site without reducing its ability to infect T cells. (d) All of the above. (e) None of the above.

soluble CD4

Ping Zhu, Ken Roux, Univ. Florida