T-Lymphocyte Gene Transfer for a Patient with ADA-SCID

1. T-Lymphocyte Gene Transfer for a Patient with ADA-SCID Onodera, M., T. Ariga, N. Kawamura, I. Koboyashi, M. Ohtsu, M. Yamada, A. Tame, H. Furuta, M. Okano, S. Matsumoto, H. Kotani, G.J. McGarrity, R. M. Blaese, and Y. Sakiyama. 1998. Blood. By: Anne Shields

2. What is ADA-SCID? • ADA- Adenosine deaminase • ADA is an enzyme • Deficiency impairs immunity responses • Resulting in SCID (Severe Combined Immunodeficiency) • Lack of enzyme of ADA coded for by a gene on chromosome 20 • Causes B and T lymphocytes to function improperly and involved in aggamglobulinea • Believed to be an autosomal recessive trait

3. ADA deficiency results in two things • Amount of circulating T lymphocytes decreases due to a high amount of toxic metabolites circulating through the body affecting the thymus • Survival rate of T cells produced in vivo decreases

4. Symptoms of ADA-SCID • Children tend to get re-occuring infections due to their low levels of immune response • Immunity responses are low because of the decrease in T lymphocytes and this leads to SCID (severe combined immunedeficiency)

5. SCID • SCID is usually a result of ADA • Congenital disease when both the humoral and cell mediated immunity is handicapped • Also associated with leukopenia and very low or non apparent levels of antibodies • SCID is either X-linked or autosomal recessive

6. There are various forms of treatment available • Bone marrow transplant • Gene therapy • Polyethylene glycol (PEG-ADA) • This article focuses on the gene therapy and bone marrow transplants

7. How can this disease be diagnosed? • There are two ways in which in can be diagnosed • Cells of the baby can be taken and tested before birth • Blood tests can also be done to determine the amounts of ADA enzyme present

9. ADA • As mentioned before, ADA is an enzyme and it is involved in the purine salvage pathway • Purine salvage pathway is involved in deamination of adenosine and deoxyadenosine and also in the formation of inosine and deoxyinosine • Patients can range from dying from this disease to having the partial ADA deficiency

10. Treatments of choice • HLA (human leukocyte antigen identical)-matched bone marrow transplant- this lets the patients to grow healthy T cell lymphocyte cells so they won’t be so susceptible to infections (recovery rates for HLA are approx 95-100%) • However, this treatment is not available to all, since there must be a compatible donor • PEG-ADA- patients get infused with purified bovine ADA that is covalently linked to PEG. PEG extends the life of ADA and reduces its ability to promote an immune response

11. What is the rationale of this paper? • Molecular analysis of a patient with ADA-SCID that was enrolled in the gene therapy protocol and his clinical results were analyzed for 18 months

12. What methods were carried out for this experiment? • Cell cultures • Patients ADA cDNA and genomic DNA were sequenced and analyzed • Southern blot analysis • Retroviral mediated gene transfer into patient’s peripheral T cells • Analysis of proviral genome • TLC of ADA activity

13. Cell cultures • B-lymphoblastoid cell lines (B-LCL) were set up

14. Sequence of ADA cDNA and genomic DNA • Total cellular RNA is isolated from B-LCL

15. Southern blot analysis • DNA was digested, separated in agarose gel, and filtered onto a nylon membrane • Filters were radioactively labeled with 32P 444bp RsaI-Pst fragment from the ADA cDNA

16. Retroviral gene transfer into peripheral T cells • T lymphoytes obtained • Placed in a medium • After 72 hours half medium was removed and replaced with retroviral vector containing interlueking-2 (IL-2) • Two transductions took place then supernatant replaced with medium containing simply IL-2( let sit for 6 days) • 11th day cells were removed, washed with saline containing 5% human albumin and injected into the patient

17. Analysis of proviral genome • Sense and anti-sense strand primers set up to corrolate to starting site (exon 7) and the eding site (exon 8) • DNA was amplified and 2 badns appeared • Transduced vs untransduced cells were compared and the ratio amplified ADA cDNA and amplified genome were calculated upon adding a probe

18. TLC of enzyme activity • Cells with one nucleus were washed with phosphate buffed saline and placed into 1% bovine serum albumin • ADA activity assayed by their conversion of 14C adenosine to 14C inosine and 14C hypoxanthine

19. Symptoms of ADA-SCID in the patient • Productive cough and Nasal Discharge • Lymphopenia • Very little mature T and B lymphocytes • Low Ig serum levels • Undetectable isohemagglutinins • No T-cell response to phytohemagglutinins, Concanavalin A, and pokeweed mitogen.

20. Mutations responsible for ADA deficiency • G632 to A transition (Arginine replaced by Histidine) • G-A transition means Guanine was reaplaced with Adenine which causes a different amino acid to be expressed( Not a silent mutation) • RsaI digestion same as father Paternal missense mutation

21. Codon Table

22. Clinical Course After Gene Therapy • 10 infusions and patient had an increase in lymphocyte numbers. Eventually returned to basal levels • PBL count remained in a normal range • Increase in CD8+ count • ADA enzyme activity increased • Immune functions improved • Ig levels increased

23. Conclusion • ADA-SCID patients had positive outcomes with the genetic therapy • Genetic therapy has established a new form of treatment for people with inherited diseases and acquired diseases