1. Safety assessment of intradiscal gene transfer: a pilot study Corey 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, MD University of Pittsburgh Medical Center, Pittsburgh, PA Presented by: David A. Ryan

2. Question What 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 What 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

3. Background Conservative and Surgical treatments often effective at relieving symptoms of degenerative disc disease Conservative and Surgical treatments often effective at relieving symptoms of degenerative disc disease –Limitations include cost and complications Target clinical symptoms instead of looking at the biological changes Target clinical symptoms instead of looking at the biological changes

4. Background Decrease in Proteoglycan (PG) concentration integral part of disc degeneration Decrease in Proteoglycan (PG) concentration integral part of disc degeneration Recent studies have successfully altered PG concentration by increasing synthesis or slowing degradation Recent studies have successfully altered PG concentration by increasing synthesis or slowing degradation –Practical application based on safety and risk analysis

5. Gene Therapy Genes give directions on how to “make” proteins Genes give directions on how to “make” proteins If genes are altered, proteins may not be made correctly  genetic disorders If genes are altered, proteins may not be made correctly  genetic disorders Can also “turn on” genes so that certain proteins are made Can also “turn on” genes so that certain proteins are made http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

6. Gene Therapy cont.. Virus used to infect cell and introduce their DNA into nucleus of cell Virus used to infect cell and introduce their DNA into nucleus of cell If successful, cell will make protein If successful, cell will make protein Body can have immune response to virus Body can have immune response to virus –Jessie Gelsinger http://ghr.nlm.nih.gov/

7. Background Early Gene Therapy focused on delivering cDNA of growth factors, ie: Transforming Growth Factor  1 (TGF-  1) Early Gene Therapy focused on delivering cDNA of growth factors, ie: Transforming Growth Factor  1 (TGF-  1) –TGF-  1 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 rejection –Done under very specific conditions Other studies assessed Bone Morphogenic Protein 2 (BMP-2) Other studies assessed Bone Morphogenic Protein 2 (BMP-2) –BMP-2 is a protein that the formation of bone and cartilage –BMP-2 is a protein that induces the formation of bone and cartilage –belongs to transforming growth factor beta (TGF-  ) family –Also demonstrated increase in PG synthesis

8. Background TGF-  1 may be harmful to several organ systems TGF-  1 may be harmful to several organ systems –Bone formation and joint scarring –Over expression can lead to immune suppression in CNS Essential to assess effects on Gene Therapy when done in close proximity to disc Essential to assess effects on Gene Therapy when done in close proximity to disc

9. Aim Assess safety of adenoviral delivery of TGF-  1 to the spinal column Assess safety of adenoviral delivery of TGF-  1 to the spinal column Explore side-effects of excessively dosed or misdirected gene therapy Explore side-effects of excessively dosed or misdirected gene therapy

10. Materials and Methods 14 NZ White Rabbits 7-9 months, 5kg 14 NZ White Rabbits 7-9 months, 5kg

11. Materials and Methods Injection Prep Injection Prep –Hanks Balanced Salt Saline (HBSS) –TGF-  1 mixed immediately before injection –Gene therapy solutions prepared immediately before injection Surgical Procedure Surgical Procedure –Animals anesthetized and prepared for sterile surgery –Incision down the center of back to reveal spinal column –Partial laminotomy (removal of bony layer) at L4 for visualization –Injection with 25 gauge needle beneath dura (outer covering) of spinal cord –Wounds were sutured close

12. Materials and Methods Post-injection Post-injection –Monitored for abnormality –Euthanized between 3 and 7 weeks –Histological analysis of spinal cord Histology Histology –Placed in 10% formalin –Section into 3-4 mm blocks within 72 hours –Processed and sectioned into (8) 7-um slices –Stained w/ hematolxylin and eosin –Examined by neuro-pathologists

13. Results Clinical Clinical –Neurological deficits found with higher concentration of TGF-  1 (paralysis) Histology Histology –No evidence of immune response in normal animals –Marked abnormalities seen in group w/ neurological deficits

14. Results

15. Results

16. Results

17. Discussion Paralysis with High conc. of TGF-  1 unexpected Paralysis with High conc. of TGF-  1 unexpected –Low concentration success promising Histological changes show proliferative response to anabolic factor AND immune response of host Histological changes show proliferative response to anabolic factor AND immune response of host Additional studies should clarify mechanism of response Additional studies should clarify mechanism of response

18. Discussion Neurological dysfunction Neurological dysfunction –Over-expression of TGF-  1 –Viral vector had immunogenic effect on spinal cord –Synergistic effect of the previous two resulted in toxicity

19. Discussion Complications not due to presence of adenoviral vector Complications not due to presence of adenoviral vector –No complications with high concentration of BMP-2 Important Important –Pathologic changes not due to basic delivery method –Different safety profiles of two factors  Future studies should look at toxicity of other growth factors –Tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) –Insulin-like growth factor-1 (IGF-1)

20. Discussion Pilot study validated use of animal model for determining toxicity of gene therapy Pilot study validated use of animal model for determining toxicity of gene therapy Limitations Limitations –Limited study- 7 weeks –Adenovirus delivery

21. Future Work Toxicity of other viral vectors Toxicity of other viral vectors Optimal experimental time frame Optimal experimental time frame Lower limit of viral concentration Lower limit of viral concentration

22. Take Home Message This Study demonstrates that misguided gene therapy may result in significant neurological changes This Study demonstrates that misguided gene therapy may result in significant neurological changes Intradural delivery of TGF-  1 at effective doses does not result in clinical or histological changes Intradural delivery of TGF-  1 at effective doses does not result in clinical or histological changes Safety Window exists for gene therapy Safety Window exists for gene therapy

23. Summary Good insight into side effects of gene therapy Good insight into side effects of gene therapy Test groups- how did they choose 14?? Test groups- how did they choose 14?? What can we learn from this? What can we learn from this?