Future Directions in Salivary Gland Research

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


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


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


Major Scientific AdvancesMajor Scientific Advances

Tools of molecular Tools of molecular biologybiology– Reverse Reverse

transcriptasetranscriptase– Restriction Restriction

endonucleasesendonucleases


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


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


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


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


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


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


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


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


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


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



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


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


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


Figure 5Figure 5


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)



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


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


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.


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.

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Future Directions in Salivary Gland Research