Download Safety assessment of intradiscal gene transfer: a pilot study

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
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
Background

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
Background
Decrease in Proteoglycan (PG)
concentration integral part of disc
degeneration
 Recent studies have successfully altered
PG concentration by increasing synthesis
or slowing degradation

– Practical application based on safety and risk
analysis
Gene Therapy

Genes 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 made
http://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 cell
 If successful, cell
will make protein
 Body can have
immune response
to virus

– Jessie Gelsinger
http://ghr.nlm.nih.gov/
Background

Early Gene Therapy focused on delivering cDNA of
growth factors, ie: Transforming Growth Factor b1 (TGFb1)
– 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 rejection
– Done under very specific conditions

Other 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) family
– Also demonstrated increase in PG synthesis
Background

TGF-b1 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
Aim

Assess safety of adenoviral delivery of
TGF-b1 to the spinal column

Explore side-effects of excessively dosed
or misdirected gene therapy
Materials and Methods

14 NZ White Rabbits
7-9 months, 5kg
Materials and Methods

Injection Prep
– Hanks Balanced Salt Saline (HBSS)
– TGF-b1 mixed immediately before injection
– Gene therapy solutions prepared immediately before injection

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
Materials and Methods

Post-injection
– Monitored for abnormality
– Euthanized between 3 and 7 weeks
– Histological analysis of spinal cord

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
Results

Clinical
– Neurological deficits found with higher
concentration of TGF-b1 (paralysis)

Histology
– No evidence of immune response in normal
animals
– Marked abnormalities seen in group w/
neurological deficits
Results
Results
Results
Discussion

Paralysis with High conc. of TGF-b1
unexpected
– Low concentration success promising
Histological changes show proliferative
response to anabolic factor AND immune
response of host
 Additional studies should clarify
mechanism of response

Discussion

Neurological dysfunction
– Over-expression of TGF-b1
– Viral vector had immunogenic effect on spinal
cord
– Synergistic effect of the previous two resulted
in toxicity
Discussion

Complications not due to presence of adenoviral
vector
– No complications with high concentration of BMP-2

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)
Discussion
Pilot study validated use of animal model
for determining toxicity of gene therapy
 Limitations

– Limited study- 7 weeks
– Adenovirus delivery
Future Work
Toxicity of other viral vectors
 Optimal experimental time frame
 Lower limit of viral concentration

Take Home Message
This Study demonstrates that misguided
gene therapy may result in significant
neurological changes
 Intradural delivery of TGF-b1 at effective
doses does not result in clinical or
histological changes
 Safety Window exists for gene therapy

Summary
Good insight into side effects of gene
therapy
 Test groups- how did they choose 14??
 What can we learn from this?
