TLymphocyte Gene Transfer for a Patient with ADASCID

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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

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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

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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

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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)

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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

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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

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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

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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

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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
  wont 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

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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

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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 patients
  peripheral T cells
• Analysis of proviral genome
• TLC of ADA activity

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Cell cultures

• B-lymphoblastoid cell lines (B-LCL) were set up

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Sequence of ADA cDNA and genomic DNA

• Total cellular RNA is isolated from B-LCL

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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

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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

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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

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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

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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.

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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

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Codon Table
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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

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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