future directions in salivary gland research
DESCRIPTION
Future Directions in Salivary Gland Research. Dennis E. Lopatin, Ph.D. Department of Biologic and Materials Sciences University of Michigan. The Impact of Gene Therapy on Dentistry. Commonly refers to any clinical application of the transfer of a foreign gene - PowerPoint PPT PresentationTRANSCRIPT
Slide No. Slide No. 11 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Future Directions in Salivary Future Directions in Salivary Gland ResearchGland Research
Dennis E. Lopatin, Ph.D.Dennis E. Lopatin, Ph.D.
Department of Biologic and Materials SciencesDepartment of Biologic and Materials Sciences
University of MichiganUniversity of Michigan
Slide No. Slide No. 22 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
The Impact of Gene Therapy The Impact of Gene Therapy on Dentistryon Dentistry
Commonly refers to any clinical application Commonly refers to any clinical application of the transfer of a foreign geneof the transfer of a foreign gene
Initially, gene therapy was associated with Initially, gene therapy was associated with either the correction of inherited genetic either the correction of inherited genetic disorders or the treatment of life-threatening disorders or the treatment of life-threatening conditionsconditions
Slide No. Slide No. 33 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..Table 1Table 1
Slide No. Slide No. 44 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
The Development of a ScienceThe Development of a Science
Gene transfer is possible due to incredible Gene transfer is possible due to incredible progress in molecular biologyprogress in molecular biology
Seminal advances in past 50 years shown in Seminal advances in past 50 years shown in next slidenext slide
Slide No. Slide No. 55 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Major Scientific AdvancesMajor Scientific Advances
Tools of molecular Tools of molecular biologybiology– Reverse Reverse
transcriptasetranscriptase– Restriction Restriction
endonucleasesendonucleases
Slide No. Slide No. 66 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
General Principles of Gene General Principles of Gene TransferTransfer
Typical mammalian geneTypical mammalian gene Many modular elementsMany modular elements
– Coding regionsCoding regions– Regulatory elementsRegulatory elements
Enzymatic tools enable researcher to modify Enzymatic tools enable researcher to modify and rearrange elementsand rearrange elements
Slide No. Slide No. 77 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Major Technological ChallengesMajor Technological Challenges
Designing the correct genetic architectureDesigning the correct genetic architecture Choice of promoter is critical for obtaining Choice of promoter is critical for obtaining
stable, high level expression of a foreign genestable, high level expression of a foreign gene Early experiments in gene transfer employed Early experiments in gene transfer employed
viral promoters that acted promiscuouslyviral promoters that acted promiscuously Not all promoters are equalNot all promoters are equal Current promoters are tissue-specific, more Current promoters are tissue-specific, more
stable gene expressionstable gene expression
Slide No. Slide No. 88 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Methods of Gene TransferMethods of Gene Transfer
Two general methods of gene transfer Two general methods of gene transfer into cellsinto cells– ViralViral– Non-viralNon-viral
Slide No. Slide No. 99 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Viral MethodsViral Methods
Greater safety riskGreater safety risk Nature’s way of efficiently transferring genesNature’s way of efficiently transferring genes Many viruses could be used. Only a few are Many viruses could be used. Only a few are
actually employedactually employed– retrovirusesretroviruses– Adeno-associated virusesAdeno-associated viruses– herpesvirusesherpesviruses– Selection criteria: tissue target, desired stability of Selection criteria: tissue target, desired stability of
gene expression, size of genegene expression, size of gene
Slide No. Slide No. 1010 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Non-Viral MethodsNon-Viral Methods SafetySafety Less efficient mechanisms for gene transferLess efficient mechanisms for gene transfer Two promising methodsTwo promising methods
– Liposomes (bags of lipid containing DNA)Liposomes (bags of lipid containing DNA)– Macromolecular conjugates (negatively charged DNA Macromolecular conjugates (negatively charged DNA
mixed with large positively charged molecules linked to a mixed with large positively charged molecules linked to a specific cell ligand)specific cell ligand)
Capable of transferring large genes, but expression is Capable of transferring large genes, but expression is transienttransient
Less risk for inflammatory or immune reactionsLess risk for inflammatory or immune reactions
Slide No. Slide No. 1111 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..Table 2Table 2
Slide No. Slide No. 1212 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Uses of Gene TransferUses of Gene Transfer Two clinical applicationsTwo clinical applications
– TherapyTherapy» Correction of an inherited or acquired defectCorrection of an inherited or acquired defect
– TherapeuticsTherapeutics» Production of biomolecules with pharmacologic Production of biomolecules with pharmacologic
functionsfunctions
Gene transfer can be accomplished two waysGene transfer can be accomplished two ways– In vivoIn vivo– Ex vivoEx vivo
Slide No. Slide No. 1313 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Applying Gene Therapy to Applying Gene Therapy to Oral CancerOral Cancer
Gene therapy for treatment of oral cancer and Gene therapy for treatment of oral cancer and precancerous lesions (E.J. Shillitoe, Univ. precancerous lesions (E.J. Shillitoe, Univ. Texas Dental Branch)Texas Dental Branch)
Reasoned that therapy is likely to be more Reasoned that therapy is likely to be more effective focused on targets expressed only in effective focused on targets expressed only in cancer cellscancer cells
Targeted human papillomaviruses, present in Targeted human papillomaviruses, present in many oral neoplasms many oral neoplasms
Slide No. Slide No. 1414 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Human Papilloma VirusesHuman Papilloma Viruses DNA viruses with an affinity for epitheliumDNA viruses with an affinity for epithelium HPV types 16 and 18 can transform normal HPV types 16 and 18 can transform normal
keratinocytes keratinocytes in vitroin vitro into an immortal, into an immortal, malignant-like phenotypemalignant-like phenotype
Requires expression of two HPV genesRequires expression of two HPV genes– E6 and E7E6 and E7
Other factors such as trauma, or an Other factors such as trauma, or an environmental irritant is needed for tumor environmental irritant is needed for tumor developmentdevelopment
Slide No. Slide No. 1515 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
HPV Gene Therapy StrategyHPV Gene Therapy Strategy Used molecules call ribozymes to disrupt Used molecules call ribozymes to disrupt
function of E6/E7function of E6/E7 Ribozymes are a class of RNA molecules that Ribozymes are a class of RNA molecules that
can act as enzymescan act as enzymes– Cleave the RNA molecules at defined sitesCleave the RNA molecules at defined sites– Cut mRNA transcripts of E6/E7Cut mRNA transcripts of E6/E7
No message, no proteinNo message, no protein Recently placed DNA encoding ribozymes in a Recently placed DNA encoding ribozymes in a
replication-deficient adenovirus vectorreplication-deficient adenovirus vector
Slide No. Slide No. 1616 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..Figure 3Figure 3
Slide No. Slide No. 1717 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Gene Transfer to Oral Gene Transfer to Oral Mucosal KeratinocytesMucosal Keratinocytes
Studies led by L.B. Taichman (Dept. of Oral Studies led by L.B. Taichman (Dept. of Oral Biology and Pathology, SUNY at Stony Brook)Biology and Pathology, SUNY at Stony Brook)
Grow keratinocytes in sheets Grow keratinocytes in sheets in vitroin vitro and return to and return to donor (eg. burn patients)donor (eg. burn patients)
No specific oral disease targeted yet, but method No specific oral disease targeted yet, but method has considerable promisehas considerable promise
Process on next slide used to transfer foreign Process on next slide used to transfer foreign genes into both epidermal and oral keratinocytesgenes into both epidermal and oral keratinocytes
Slide No. Slide No. 1818 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..Figure 4Figure 4
Slide No. Slide No. 1919 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Gene Transfer to Salivary Gene Transfer to Salivary Glands (NIDR)Glands (NIDR)
Bruce J. Baum and Brian C. O’ConnellBruce J. Baum and Brian C. O’Connell Easy target for Easy target for in vivoin vivo gene transfer because of gene transfer because of
anatomic locationanatomic location Initial studies examined feasibility of using Initial studies examined feasibility of using
replication-deficient recombinant adenovirus vectors replication-deficient recombinant adenovirus vectors to transfer foreign genes into rat salivary glands to transfer foreign genes into rat salivary glands in in vivovivo
Slide No. Slide No. 2020 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Gene Transfer into Rat Gene Transfer into Rat Salivary Glands (con’td.)Salivary Glands (con’td.)
Vectors could infect Vectors could infect in vitroin vitro Administered vectors to cannulated ducts through the Administered vectors to cannulated ducts through the
duct orifice via retrograde injectionduct orifice via retrograde injection All major rat salivary glands (parotid, submandibular, All major rat salivary glands (parotid, submandibular,
and sublingual) could be infected by adenovirusesand sublingual) could be infected by adenoviruses Histology showed that both acinar and ductal Histology showed that both acinar and ductal
epithelial cells could act as recipients for gene transferepithelial cells could act as recipients for gene transfer
Slide No. Slide No. 2121 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Repair of Irreversibly Damaged Repair of Irreversibly Damaged Acinar CellsAcinar Cells
Two situations result in acinar cell damageTwo situations result in acinar cell damage– Therapeutic irradiation of head and neckTherapeutic irradiation of head and neck– Sjögren’s syndrome (an autoimmune exocrinopathy)Sjögren’s syndrome (an autoimmune exocrinopathy)
Goal was to convert surviving ductal cells into acinar-Goal was to convert surviving ductal cells into acinar-like cells that secrete salt and fluidlike cells that secrete salt and fluid
An example of “organ engineering”: Changing the An example of “organ engineering”: Changing the basic function of a cell typebasic function of a cell type
Adenoviral-mediated transfer of aquaporin-1 into rat Adenoviral-mediated transfer of aquaporin-1 into rat salivary glandsalivary gland
Slide No. Slide No. 2222 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Figure 5Figure 5
Slide No. Slide No. 2323 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Gene TherapeuticsGene Therapeutics
Use normally functioning salivary gland to deliver Use normally functioning salivary gland to deliver biopharmaceuticalsbiopharmaceuticals
Feasibility has been demonstrated by transferring Feasibility has been demonstrated by transferring in in vivovivo, gene for human , gene for human 1-antitrypsin (liver protein) into 1-antitrypsin (liver protein) into rat submandibular glands.rat submandibular glands.
Other candidates: histatin, Other candidates: histatin, P. gingivalisP. gingivalis fimbrillin (local fimbrillin (local immunization to make sIgA)immunization to make sIgA)
Slide No. Slide No. 2424 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..Figure 6Figure 6
Slide No. Slide No. 2525 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
The Future of Gene Transfer The Future of Gene Transfer and its Impact on Dentistryand its Impact on Dentistry
Now accepted a feasible by the general Now accepted a feasible by the general biomedical communitybiomedical community
No longer considered an esoteric exercise with No longer considered an esoteric exercise with practical applicationpractical application
Numerous articles on gene transfer in Numerous articles on gene transfer in mainstream journals (e.g.., NEJM).mainstream journals (e.g.., NEJM).
Not a panacea for all clinical problemsNot a panacea for all clinical problems
Slide No. Slide No. 2626 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Current Tools are CrudeCurrent Tools are Crude Vectors available for gene transfer have problemsVectors available for gene transfer have problems
– Transient and inflammatory nature of adenovirus useTransient and inflammatory nature of adenovirus use– Low titersLow titers– Mutagenic potential (safety concerns)Mutagenic potential (safety concerns)
Biotechnical Industry is addressing shortcomingsBiotechnical Industry is addressing shortcomings– Tremendous commercial potentialTremendous commercial potential
Treatments appear heroic, mechanics of gene Treatments appear heroic, mechanics of gene transfer are mundanetransfer are mundane
Slide No. Slide No. 2727 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
ConclusionConclusion Initially, gene transfer approaches will not be used for Initially, gene transfer approaches will not be used for
routine careroutine care– Refractory to conventional treatment (high risk for Refractory to conventional treatment (high risk for
periodontal disease or caries)periodontal disease or caries) Envision scenarios in which gene transfer is applied Envision scenarios in which gene transfer is applied
to periodontal bone loss, oral ulcers, delayed tooth to periodontal bone loss, oral ulcers, delayed tooth eruptioneruption
Biology is changing rapidly and will dramatically Biology is changing rapidly and will dramatically impact on the way dentistry is practiced.impact on the way dentistry is practiced.
Slide No. Slide No. 2828 Dennis E. Lopatin, Ph.D..Dennis E. Lopatin, Ph.D..
Excellent ReferencesExcellent References
Baum, BJ and O’Connell, BC. The Impact of Baum, BJ and O’Connell, BC. The Impact of Gene Therapy on Dentistry. Gene Therapy on Dentistry. JADAJADA, , 126126:179-189, :179-189, 1995.1995.
Delporte, C, O’Connell, BC, He, X, Lancaster, Delporte, C, O’Connell, BC, He, X, Lancaster, HE, O’Connell, AC, Agre, P, and Baum, BJ. HE, O’Connell, AC, Agre, P, and Baum, BJ. Increased fluid secretion after adenoviral-Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. irradiated rat salivary glands. PNASPNAS, , 9494:3268-:3268-3273, 1997.3273, 1997.