Safety assessment of intradiscal gene transfer: a pilot studyCorey J. Wallach, MD, Joesph S. Kim, MD, Satoshi Sobajima, MD, Christian Lattermann, MD, William M. Oxner, MD, Kathryn McFadden, MD, Paul D. Robbins, PhD, Lars G. Gilbertson, PhD, James D. Kang, MDUniversity of Pittsburgh Medical Center, Pittsburgh, PA

Presented by:David A. Ryan

QuestionWhat are the potential side effects of excessively dosed or misdirected Gene Therapy with adenoviral delivery TGF-B1 compared to delivery of recombinant protein TGF-1 and BMP-2

BackgroundConservative and Surgical treatments often effective at relieving symptoms of degenerative disc diseaseLimitations include cost and complicationsTarget clinical symptoms instead of looking at the biological changes

BackgroundDecrease in Proteoglycan (PG) concentration integral part of disc degenerationRecent studies have successfully altered PG concentration by increasing synthesis or slowing degradationPractical application based on safety and risk analysis

Gene TherapyGenes give directions on how to make proteins

If genes are altered, proteins may not be made correctly genetic disorders

Can also turn on genes so that certain proteins are madehttp://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

Gene Therapy cont..Virus used to infect cell and introduce their DNA into nucleus of cellIf successful, cell will make proteinBody can have immune response to virusJessie Gelsingerhttp://ghr.nlm.nih.gov/

BackgroundEarly Gene Therapy focused on delivering cDNA of growth factors, ie: Transforming Growth Factor b1 (TGF-b1)TGF-b1 is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types Demonstrated beneficial effect w/o evidence of immune response or host rejectionDone under very specific conditionsOther studies assessed Bone Morphogenic Protein 2 (BMP-2)BMP-2 is a protein that induces the formation of bone and cartilage belongs to transforming growth factor beta (TGF-b) familyAlso demonstrated increase in PG synthesis

BackgroundTGF-b1 may be harmful to several organ systemsBone formation and joint scarringOver expression can lead to immune suppression in CNSEssential to assess effects on Gene Therapy when done in close proximity to disc

AimAssess safety of adenoviral delivery of TGF-b1 to the spinal column

Explore side-effects of excessively dosed or misdirected gene therapy

Materials and Methods14 NZ White Rabbits 7-9 months, 5kg

Materials and MethodsInjection PrepHanks Balanced Salt Saline (HBSS)TGF-b1 mixed immediately before injectionGene therapy solutions prepared immediately before injection

Surgical ProcedureAnimals anesthetized and prepared for sterile surgeryIncision down the center of back to reveal spinal columnPartial laminotomy (removal of bony layer) at L4 for visualizationInjection with 25 gauge needle beneath dura (outer covering) of spinal cordWounds were sutured close

Materials and MethodsPost-injectionMonitored for abnormalityEuthanized between 3 and 7 weeksHistological analysis of spinal cordHistologyPlaced in 10% formalinSection into 3-4 mm blocks within 72 hoursProcessed and sectioned into (8) 7-um slicesStained w/ hematolxylin and eosinExamined by neuro-pathologists

ResultsClinicalNeurological deficits found with higher concentration of TGF-b1 (paralysis)HistologyNo evidence of immune response in normal animalsMarked abnormalities seen in group w/ neurological deficits

Results

Results

Results

DiscussionParalysis with High conc. of TGF-b1 unexpectedLow concentration success promisingHistological changes show proliferative response to anabolic factor AND immune response of hostAdditional studies should clarify mechanism of response

DiscussionNeurological dysfunctionOver-expression of TGF-b1Viral vector had immunogenic effect on spinal cordSynergistic effect of the previous two resulted in toxicity

DiscussionComplications not due to presence of adenoviral vectorNo complications with high concentration of BMP-2

ImportantPathologic changes not due to basic delivery methodDifferent safety profiles of two factors Future studies should look at toxicity of other growth factorsTissue inhibitor of matrix metalloproteinases-1 (TIMP-1)Insulin-like growth factor-1 (IGF-1)

DiscussionPilot study validated use of animal model for determining toxicity of gene therapyLimitationsLimited study- 7 weeksAdenovirus delivery

Future WorkToxicity of other viral vectorsOptimal experimental time frameLower limit of viral concentration

Take Home MessageThis Study demonstrates that misguided gene therapy may result in significant neurological changesIntradural delivery of TGF-b1 at effective doses does not result in clinical or histological changesSafety Window exists for gene therapy

SummaryGood insight into side effects of gene therapyTest groups- how did they choose 14??What can we learn from this?