Safety assessment of Safety assessment of intradiscal gene intradiscal gene

transfer: a pilot studytransfer: a pilot studyCorey J. Wallach, MD, Joesph S. Kim, MD, Satoshi Sobajima, MD, Corey J. Wallach, MD, Joesph S. Kim, MD, Satoshi Sobajima, MD,

Christian Lattermann, MD, William M. Oxner, MD, Kathryn Christian Lattermann, MD, William M. Oxner, MD, Kathryn McFadden, MD, Paul D. Robbins, PhD, Lars G. Gilbertson, PhD, McFadden, MD, Paul D. Robbins, PhD, Lars G. Gilbertson, PhD,

James D. Kang, MDJames D. Kang, MDUniversity of Pittsburgh Medical Center, Pittsburgh, PAUniversity of Pittsburgh Medical Center, Pittsburgh, PA

Presented by:Presented by:David A. RyanDavid A. Ryan

QuestionQuestion

What are the potential side effects of What are the potential side effects of excessively dosed or misdirected excessively dosed or misdirected Gene Therapy with adenoviral Gene Therapy with adenoviral delivery TGF-B1 compared to delivery TGF-B1 compared to delivery of recombinant protein TGF-delivery of recombinant protein TGF-1 and BMP-21 and BMP-2

BackgroundBackground

Conservative and Surgical Conservative and Surgical treatments often effective at treatments often effective at relieving symptoms of degenerative relieving symptoms of degenerative disc diseasedisc disease– Limitations include cost and Limitations include cost and

complicationscomplications Target clinical symptoms instead of Target clinical symptoms instead of

looking at the biological changes looking at the biological changes

BackgroundBackground

Decrease in Proteoglycan (PG) Decrease in Proteoglycan (PG) concentration integral part of disc concentration integral part of disc degenerationdegeneration

Recent studies have successfully Recent studies have successfully altered PG concentration by altered PG concentration by increasing synthesis or slowing increasing synthesis or slowing degradationdegradation– Practical application based on safety Practical application based on safety

and risk analysisand risk analysis

Gene TherapyGene Therapy

Genes give directions on how to Genes give directions on how to “make” proteins“make” proteins

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

Can also “turn on” genes so that Can also “turn on” genes so that certain proteins are madecertain proteins are made

http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

Gene Therapy cont..Gene Therapy cont.. Virus used to Virus used to

infect cell and infect cell and introduce their introduce their DNA into nucleus DNA into nucleus of cellof cell

If successful, cell If successful, cell will make proteinwill make protein

Body can have Body can have immune immune response to virusresponse to virus– Jessie GelsingerJessie Gelsinger

http://ghr.nlm.nih.gov/

BackgroundBackground Early Gene Therapy focused on delivering cDNA of Early Gene Therapy focused on delivering cDNA of

growth factors, ie: Transforming Growth Factor growth factors, ie: Transforming Growth Factor 1 1 (TGF-(TGF-1)1)– TGF-TGF-1 is a multifunctional peptide that controls 1 is a multifunctional peptide that controls

proliferation, differentiation, and other functions in many proliferation, differentiation, and other functions in many cell types cell types

– Demonstrated beneficial effect w/o evidence of immune Demonstrated beneficial effect w/o evidence of immune response or host rejectionresponse or host rejection

– Done under very specific conditionsDone under very specific conditions Other studies assessed Bone Morphogenic Protein Other studies assessed Bone Morphogenic Protein

2 (BMP-2)2 (BMP-2)– BMP-2 is a protein that BMP-2 is a protein that induces the formation of bone the formation of bone

and cartilage and cartilage – belongs to transforming growth factor beta (TGF-belongs to transforming growth factor beta (TGF-) )

familyfamily– Also demonstrated increase in PG synthesisAlso demonstrated increase in PG synthesis

BackgroundBackground

TGF-TGF-1 may be harmful to several 1 may be harmful to several organ systemsorgan systems– Bone formation and joint scarringBone formation and joint scarring– Over expression can lead to immune Over expression can lead to immune

suppression in CNSsuppression in CNS Essential to assess effects on Gene Essential to assess effects on Gene

Therapy when done in close Therapy when done in close proximity to discproximity to disc

AimAim

Assess safety of adenoviral delivery Assess safety of adenoviral delivery of TGF-of TGF-1 to the spinal column1 to the spinal column

Explore side-effects of excessively Explore side-effects of excessively dosed or misdirected gene therapydosed or misdirected gene therapy

Materials and MethodsMaterials and Methods

14 NZ White 14 NZ White Rabbits 7-9 Rabbits 7-9 months, 5kgmonths, 5kg

Materials and MethodsMaterials and Methods Injection PrepInjection Prep

– Hanks Balanced Salt Saline (HBSS)Hanks Balanced Salt Saline (HBSS)– TGF-TGF-1 mixed immediately before injection1 mixed immediately before injection– Gene therapy solutions prepared immediately before Gene therapy solutions prepared immediately before

injectioninjection

Surgical ProcedureSurgical Procedure– Animals anesthetized and prepared for sterile surgeryAnimals anesthetized and prepared for sterile surgery– Incision down the center of back to reveal spinal columnIncision down the center of back to reveal spinal column– Partial laminotomy (removal of bony layer) at L4 for Partial laminotomy (removal of bony layer) at L4 for

visualizationvisualization– Injection with 25 gauge needle beneath dura (outer Injection with 25 gauge needle beneath dura (outer

covering) of spinal cordcovering) of spinal cord– Wounds were sutured closeWounds were sutured close

Materials and MethodsMaterials and Methods

Post-injectionPost-injection– Monitored for abnormalityMonitored for abnormality– Euthanized between 3 and 7 weeksEuthanized between 3 and 7 weeks– Histological analysis of spinal cordHistological analysis of spinal cord

HistologyHistology– Placed in 10% formalinPlaced in 10% formalin– Section into 3-4 mm blocks within 72 hoursSection into 3-4 mm blocks within 72 hours– Processed and sectioned into (8) 7-um slicesProcessed and sectioned into (8) 7-um slices– Stained w/ hematolxylin and eosinStained w/ hematolxylin and eosin– Examined by neuro-pathologists Examined by neuro-pathologists

ResultsResults

ClinicalClinical– Neurological deficits found with higher Neurological deficits found with higher

concentration of TGF-concentration of TGF-1 (paralysis)1 (paralysis) HistologyHistology

– No evidence of immune response in No evidence of immune response in normal animalsnormal animals

– Marked abnormalities seen in group w/ Marked abnormalities seen in group w/ neurological deficitsneurological deficits

ResultsResults

ResultsResults

ResultsResults

DiscussionDiscussion

Paralysis with High conc. of TGF-Paralysis with High conc. of TGF-1 1 unexpectedunexpected– Low concentration success promisingLow concentration success promising

Histological changes show Histological changes show proliferative response to anabolic proliferative response to anabolic factor AND immune response of hostfactor AND immune response of host

Additional studies should clarify Additional studies should clarify mechanism of responsemechanism of response

DiscussionDiscussion

Neurological dysfunctionNeurological dysfunction– Over-expression of TGF-Over-expression of TGF-11– Viral vector had immunogenic effect on Viral vector had immunogenic effect on

spinal cordspinal cord– Synergistic effect of the previous two Synergistic effect of the previous two

resulted in toxicityresulted in toxicity

DiscussionDiscussion

Complications not due to presence of Complications not due to presence of adenoviral vectoradenoviral vector– No complications with high concentration of BMP-2 No complications with high concentration of BMP-2

ImportantImportant– Pathologic changes not due to basic delivery Pathologic changes not due to basic delivery

methodmethod– Different safety profiles of two factors Different safety profiles of two factors

Future studies should look at toxicity of other growth Future studies should look at toxicity of other growth factorsfactors

– Tissue inhibitor of matrix metalloproteinases-1 (TIMP-1)Tissue inhibitor of matrix metalloproteinases-1 (TIMP-1)– Insulin-like growth factor-1 (IGF-1)Insulin-like growth factor-1 (IGF-1)

DiscussionDiscussion

Pilot study validated use of animal Pilot study validated use of animal model for determining toxicity of model for determining toxicity of gene therapygene therapy

LimitationsLimitations– Limited study- 7 weeksLimited study- 7 weeks– Adenovirus deliveryAdenovirus delivery

Future WorkFuture Work

Toxicity of other viral vectorsToxicity of other viral vectors Optimal experimental time frameOptimal experimental time frame Lower limit of viral concentrationLower limit of viral concentration

Take Home MessageTake Home Message

This Study demonstrates that This Study demonstrates that misguided gene therapy may result misguided gene therapy may result in significant neurological changesin significant neurological changes

Intradural delivery of TGF-Intradural delivery of TGF-1 at 1 at effective doses does not result in effective doses does not result in clinical or histological changesclinical or histological changes

Safety Window exists for gene Safety Window exists for gene therapytherapy

SummarySummary

Good insight into side effects of gene Good insight into side effects of gene therapytherapy

Test groups- how did they choose Test groups- how did they choose 14??14??

What can we learn from this?What can we learn from this?