Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-04-28
2002-11-26
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S366000, C435S378000, C536S023100
Reexamination Certificate
active
06485976
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
This application relates to the field of genetic modification. In particular, the invention disclosed herein provides a method of transducing cells with cytoprotective genes using adeno-associated viral vectors. Specifically, in a preferred embodiment the invention relates to a method of preventing or reducing the rejection of grafted insulin-producing pancreatic &bgr;-cells and islets by transduction of the grafted cells with cytoprotective genes. In another preferred embodiment, the invention relates to a method of protecting pancreatic islet &bgr;-cells from immune destruction in a patient to protect against the development of Type 1 diabetes.
2. Description of the Background Art
Type 1 diabetes is an autoimmune disease that ultimately results in destruction of the insulin producing &bgr;-cells in the pancreas. In type 1 diabetes, invading cells, primarily lymphocytes and macrophages, enter the islets and release toxic substances called cytokines which in turn set off an inflammatory reaction. Cytokines and immune signals play an important role in the overall defense mechanisms of the body, but can also be released in an unregulated fashion, leading to pancreatic islet cell damage in Type 1 diabetes or destruction of transplanted islet cells. Cytokines or other immune factors may damage the islets directly by stimulating “death” signals within the insulin producing &bgr;-cells or indirectly by causing other non-&bgr;-cells to produce a milieu that is cytotoxic to &bgr;-cells. In either case, destruction of the insulin-producing &bgr;-cells follows, along with the well known sequelae of hyperglycemia.
Although patients with Type 1 diabetes may be treated adequately with insulin injections or insulin pumps, these therapies are only partially effective. Insulin replacement via insulin or pump cannot fully reverse the defect in vascular endothelium found in the hyperglycemic state. Pieper et al.,
Diabetes Res. Clin. Pract
. Suppl.:S157-S162 (1996). In addition, frequent hyper- and hypoglycemia typically occurs despite intensive home blood glucose monitoring. Finally, careful dietary constraint is needed to maintain an adequate ratio of calories consumed to insulin. This often causes major psychosocial stress for many diabetic patients. Development of methods to transplant functional pancreatic islets into diabetic patients would overcome most of these problems and result in improved life expectancy and quality of life.
The approach taken with this invention offers numerous advantages lacking in prior art therapies currently available and improves the success of known treatments such as islet cell transplantation. Currently, most treatments and therapy of diabetes focus on direct insulin replacement. Unfortunately, transplanted allo- or xenogeneic pancreatic islet cells, like whole-organ transplants, are subject to graft rejection as are other solid organ transplants.
The only viable method currently available in the prior art of preventing transplant rejection involves systemic immunosuppressive therapy, however immunosuppression can have serious, long-term effects on the graft recipient. Research aimed at the protection of transplanted allogeneic human pancreatic islets through genetic manipulation of the transplanted cells likewise has focussed on general immunosuppression. For example, Tahara et al. (
Transplantation Proc.
24(6):2975-2976 (1992)) have expressed the immunosuppressive cytokine IL-10 in cultured cells using both retroviral and adeno-associated viral vectors to resolve the problems caused by the immune response to the transplanted cells. However, this report did not provide any evidence whether adeno-associated vectors could successfully deliver genes to &bgr;-cells or to pancreatic islets.
Immune-induced islet cytotoxicity plays a significant role in both autoimmune &bgr;-cell destruction in Type 1 diabetes and acute graft rejection after &bgr;-islet transplantation. General immunosuppression methods are designed to combat this toxicity. It is known that pancreatic &bgr;-cells, whether native or transplanted, undergo inflammatory damage and cell death upon chronic exposure to cytotoxic cytokines such as interleukin-1&bgr; (IL-1&bgr;) (Dunger et al.,
J. Autoimmunity
9:309-313 (1996); Mandrup-Poulsen et al., Diabetologia 29:63-6 (1986)). Cytokine treated islets in vitro demonstrate morphologic evidence of apoptosis such as nuclear condensation, intracytoplasmic vacuole formation, mitochondrial swelling, insulin degranulation, and preservation of the cell membrane when viewed under the electron microscope. (Ling et al.,
Diabetes
42:56-65 (1993); Fehsel et al.,
Diabetes
42:496-500 (1993)). A proposed method of circumventing the rejection mechanism without systemic immunosuppression involves introducing cytoprotective genes such as immunosuppressive cytokines into the donor tissue by means of a viral vector. Methods discussed in the prior art have used retroviral (including lentiviral) and adenoviral vectors. See e.a. Tahara et al.,
Transplantation Proc.
24(6):2975-2976 (1992); Smith et al.,
Transplantation
64:1040-1049 (1997); Hayashi,
Transplantation Proc.
29:2213 (1997); Naldini,
Science
272:263-267 (1996); Xi,
Neurochem. Int.
22(5):511-516 (1993). Hayashi et al. have suggested adenovirus-mediated transduction of an antisense ribozyme for both the &agr;(1,3)-galactosyl transferase gene and the &agr;(1,2)-fucosyl transferase gene to xenogeneic cells and organs to inhibit hyperacute rejection.
For successful protection of pancreatic islets by genetic transduction, the vector used must be non-pathogenic, must be capable of stable gene expression, should not be inflammatory or cause the expression of immunogenic peptides and of course must be able to infect pancreatic &bgr;-cells. All the vector types currently proposed for transfer of genes to pancreatic &bgr;-cells lack at least one of the above properties which are desired for use in protection of pancreatic islets by genetic transduction.
Relatively few studies have used viral vectors to introduce transgenes into pancreatic islets. Csete and colleagues (
Transplantation
59(2): 263-268 (1995)) showed that adenoviral vectors could effectively transfer
E. coli
&bgr;-galactosidase (&bgr;-gal) into mouse islets for up to 10 days in culture and that islet insulin secretion was not impaired by the viral DNA. Gene expression was confirmed by the demonstration of &bgr;-gal mRNA and high levels of functional &bgr;-gal protein. After 10 days, the &bgr;-gal protein returned to pre-transfection levels, indicating that the transgene was not incorporated into the host genome. Adenovirus-mediated gene transfer also has been achieved in mouse islets using a &bgr;-gal reporter gene by Sigalla and colleagues (
Human Gene Therapy
8(13):1625-1634 (1997)). Transduced islets had normal in vitro glucose-stimulated insulin secretion and were able to normalize blood glucose when transplanted into syngeneic and allogeneic streptozotocin-induced diabetic mice.
In addition, ex vivo gene transfer into mouse islets has been successfully performed using an adenoviral vector with approximately 50% of the islets showing positive staining for &bgr;-gal which was detectable for 8 weeks (Smith et al., Transplantation 64(7):1040-1049 (1997)). Gene transfer to human pancreatic &agr;- and &bgr;-cells have also been demonstrated using adenovirus-polylysine/DNA complexes with peak levels of expression lasting for 5-7 days (Becker et al., J. Biol. Chem. 269(33):21234-21238 (1994)). There, both polycationic liposome- and adenoviral-mediated gene transfer yielded 50-70% &bgr;gal positive cells, but chloramphenicol acetyl transferase activity degenerated after 5-7 days indicating that the transgene was not incorporated into the host genome. In addition, intact human islets showed lower transduction efficiencies than dispersed islet cells, possibly due to fewer cells being exposed to virus. In any case, adenoviral vectors were not capable of long-term, stable integration of genes into the i
Bleich David
Nadler Jerry L.
Prasad Konkal-Matt R.
City of Hope
McGarry Sean
Rothwell Figg Ernst & Manbeck P.C.
Zara Jane
LandOfFree
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