The enzyme adenosine deaminase     ADA deficiency             Treatment         Sources

Adenosine deaminase (Fig. 1), or ADA (also referred to as adenosine aminohydrolase, EC 3.5.4.4), is a ubiquitous enzyme which appears to be particularly important in the development of thymocytes.  ADA converts adenosine into inosine (Fig. 2) and converts deoxyadenosine (dAdo) into deoxyinosine, both through the hydrolysis of the purine amino group (Benveniste and Cohen, 1995).  ADA is present in all tissues, but has much higher activity in lymphocyte development, perhaps due to its direct association with CD26, which is exhibited on activated T cells (Tsuboi, et al., 1995).  ADA activity is particularly high in thymocytes of the thymic cortex, but drops off rapidly in the medulla (Resta, et al., 1997).  There are two enzymes which carry out ADA activity, called ADA1 and ADA2.  ADA1 (Fig. 1), a 40 kD monomeric protein with a 200 kD, noncatalytic combining protein, is responsible for about 90% of adenosine deamination.  ADA2 is somewhat larger at 110 kD, appears to play a general adenosine deamination role in serum (Tsuboi, et al., 1995).


Figure 2:  ADA hydrolysis of adenosine into inosine.  ADA also acts on deoxyadenosine, which has no 2' hydroxy group on the ribose unit, and dATP, which also has three phospate units on the 5' hydroxy group.  Hydrolysis cleaves the amino group, resulting in an amide, and releasing an ammonia molecule.

ADA Deficiency     Adenosine deaminase             Treatment         Sources

These conversions are beginnings of important degratory pathways of purines in cells.  The main biochemical consequences are:

• Individuals that are ADA deficient have abnormal accumulations of dAdo and dATP (deoxyadenosine triphosphate), which is the phosphorylated end-product of of dAdo.
• These accumulations result in the inactivation of an enzyme called S-adenosylhomocysteine hydrolase (SAH), whose substrate accumulation inhibits certain methylations of nucleic acids, proteins, and lipids (Benveniste and Cohen, 1995).
• High levels of dAdo are also responsible for an inhibition of ribonucleotide reductase, causing an inbalance of the deoxynucleotides (dNTP).
• This imbalance, in addition to the enzyme inhibitions, leads to impairment of DNA synthesis and repair in T lymphocytes (Benveniste and Cohen, 1995).

Symptoms for ADA deficiency-induced SCID are usually immediately seen in affected infants, since the disorder is genetic, but there have been cases of mild ADA deficiency which were not been detected until older childhood and even adulthood (Ozsahin, et al., 1997).  Complete ADA deficiency results in fatal infantile onset syndrome of SCID.  Even in milder cases, T-cell function is severely depressed, and antibody responses are barely produced, resulting in a highly immunocompromised individual (Ochs, et al., 1992).  Patients with the disorder exhibit growth retardation, are susceptible to opportunistic infections, lymphopenia, and defective cellular and humoral immune responses (Ozsahin, et al., 1997).  At least 40 alleles have been identified to cause ADA deficiency.  There appear to be specific locations on the gene which are unusually succeptible to mutations in the characterized alleles, so called hot-spot mutations (Hirschhorn, et al., 1990).

It has been noted above that cortical thymocytes exhibit high levels of ADA activity at the CD8^low and CD4⁺CD8⁺ stages of development, which coincide with recombination of the T cell receptor (TCR).  One of the consequences of dAdo and dNTP imbalances at this stage is that V(D)J recombination is affected by the imbalance of nucleotides available for the non-template encoding regions, or N-regions, during assembly of the V(D)J joints of TCR chains and immunoglobulins.  N-regions are typically G-C rich, but cells treated with dAdo, which simulates ADA deficiency, exhibit N-regions with significant A-T nucelosides (Gangi-Peterson, et al., 1999).  This tendency was also found in isolated cells of ADA deficient patients, and the recombination frequency of Igs and TCRs are significantly reduced (Gangi-Peterson, et al., 1999).  The specific effects of this change are unknown, but there is potential for disruption of the V(D)J recombination event or for a decrease in the diversity of immunoglobulins and T cell receptors, which might constrain immune responses.  TdT is the enzyme that adds the N-nucleotides in recombination, and TdT knockout mice interestingly have normal B cell and T cell responses (Gangi-Peterson, et al., 1999), indicating that the A-T rich N-regions may have a more deleterious effect on development than no N-regions at all.

Treatment of ADA deficiency is possible through 3 main routes:

• Bone marrow or stem cell transplants from a haploidentical donor is available for a minority of patients (Bordignon et al., 1995).  Since the disorder is genetic, haploidentical donors who are not ADA deficient themselves are harder to find than for leukemia or another bone marrow transplant scenario.  Bone marrow transplants can be performed to correct general cases of SCID, and the new marrow cells contain the appropriate genes, including ADA⁺, to develop normal T and B cells, reconstituting immune function (Bordignon, et al., 1995; Haddad, et al. 1999).

• Enzyme therapy can directly add missing ADA.  This can occur through a transfusion of irradiated red blood cells.  Patients on this regime experienced equally depressed antibody responses as untreated ADA deficient patients (Ochs, et al., 1992).  Direct enzyme injections are a better method of introducing ADA to the patient.  There have also been several studies which have discovered efficient methods of obtaining high yield human ADA by transfecting various cells and insect larvae with human ADA cDNA (Medin, et al., 1990).  In treatment, human or bovine ADA is covalently attached to polyethylene glycol (PEG), which blocks access of degrative enzymes to ADA, lengthening the plasma half-life from a few minutes to 24 hours.  Weekly injections of “PEGilated” ADA usually reverse the main consequences of genomic ADA deficiency, as lymphocyte levels and proliferative responses to antigen normalize (Bordignon, et al., 1995).  Levels of dAdo and dATP are also reduced, as expected in an effective treatment.  Enzyme therapy does not work for all patients.  In some individuals, T cell function increases to mitogens in vitro, but the patient is not able to consistently mount immune responses to select antigens (Blaese, et al., 1995), resulting in PEG-ADA treatment failure.

• Somatic gene therapy can create functional ADA⁺ T cells.  ADA deficiency was the first disorder to be treated by gene therapy (Bordignon, et al., 1995).  The initial targets for genetic manipulation were bone marrow (BM) stem cells and peropheral blood lymphocytes (PBL).  Vectors expressing human ADA cDNA, 1.5 kbp (Blaese, et al., 1995) with their own promoters can be transfected into BM stem cells and PBLs in vitro.  6 months after gene therapy ended, vector-derived DNA is found in PBLs, and levels of leukocytes and bone marrow progenitors increases.  ADA activity is substantially improved, and short term (6-12 months) immunity is effectively restored (Bordignon, et al., 1995).  These experiments also confirm that genetically modified cells with properly functioning ADA have a selective advantage over endogenous ADA deficient cells during development. Even growth was normalized in some patients whose growth was not improved with PEG-ADA treatment (Bordignon, et al., 1995).

Since T cells suffer most from ADA deficiency, and T cells were essentially the only cells whose ADA activity rose under gene therapy, the possibility of T-cell directed gene modification was also investigated (Blaese, et al., 1995).  T cells are obtained through apheresis, and they proliferate in culture, while they are transfected with human ADA cDNA from a retrovirus.  After 9-12 days, the T cells are reintroduced into the patient (Blaese, et al., 1995).  The ability to transfect cells in vitro offers an advantage that the patient is not injected with a virus, which could have uncontrollable consequences.  The direct targeting also increases the effectiveness of even low gene transfer efficiency.  With T cells, even 1% gene transfer efficiency results in 10⁹ to 10¹⁰ new T cells, which contribute to immune diversity (Blaese, et al., 1995).

The use of gene therapy has more ethical concerns than the other methods, but there are key advantages to its use in this disorder.  First, the patient can continue PEG-ADA treatment, so that no treatment is withheld.  Second, live virus is not introduced into the patient, as was the case in the recent death of 18-year old Jesse Gelsinger, who was undergoing gene therapy for ornithine transcarbamylase deficiency (OTC), a liver disorder.  (CNN.com, December 8, 1999)  Instead, T cells are isolated, transfected, then reintroduced to the patient, avoiding any potential complications with a virus.  Third, T cells are directly targeted instead of stem cells, and they are long-lived and proliferate well after genetic manipulation. (Pollok, et al., 1998)  This makes gene therapy an attractive solution to the problems created by ADA deficiency, which can be used when enzyme therapy fails.

Sources      Adenosine deaminase             ADA deficiency             Treatment

Benveniste P, Cohen A. 1995 August 29. p53 expression is required for thymocyte apoptosis induced by adenosine deaminase deficiency. Proceedings of the National Academy of Science 92: 8373-8377. Abstract   PDF Text

Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casatori G, Panina P, Mazzolari E, Maggiono D, Rossi C, Servida P, Ugazio AG, Mavilio F. 1995 October 20. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients. Science 270: 470-475. Abstract

Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF. 1995 October 20. T lymphocyte-directed gene therapy for ADA^- SCID: Initial trial results after 4 years. Science 270: 475-480. Abstract

Gangi-Peterson L, Sorscher DH, Reynolds JW, Kepler TB, Mitchell BS. 1999 March 15. Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination. The Journal of Clinical Investigations 103: 833-841.  Abstract     Full Text

Haddad E, Le Diest F, Aucouturier P, Cavazzana-Calvo M, Blanche S, de Saint Basile G, Fischer A. 1999 October 15. Long-term chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: a single-center study of 22 patients. Blood. 94: 2923-2930.  Abstract   Full Text

Hirschhorn R, Tzall S, Ellenbogen A. 1990 August 15. Hot spot mutations in adenosine deaminase deficiency. Proceedings of the National Academy of Science 87: 6171-6175. Abstract   PDF Text

Lynch CM, Clowes MM, Osborne WRA, Clowes AW, Miller AD. 1992 February 1. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy. Proceedings of the National Academy of Science 89: 1138-1142. Abstract   PDF Text

Medin JA, Hunt L, Gathy K, Evans RK, Coleman MS. 1990 April. Efficient, low-cost protein factories: Expression of human adenosine deaminase in baculovirus-infected insect larvae. Proceedings of the National Academy of Science 87: 2760-2764.  Abstract   PDF Text

Ochs HD, Buckley RH, Kobayashi RH, Kobayashi AL, Sorensen RU, Douglas SD, Hamilton BL, Hershfield MS. 1992 September 1. Antibody responses to bacteriophage phi X174 in patients with adenosine deaminase deficiency. Blood 80: 1163-1171. Abstract

Ozsahin H, Arredondo-Vega FX, Santisteban I, Fuhrer H, Tuchschmid P, Jochum W, Aguzzi A, Lederman HM, Fleischman A, Winkelstein JA, Seger RA, Hershfield MS. 1997 April 15. Adenosine deaminase deficiency in adults. Blood 89: 2849-2855.  Abstract   Full Text

Pollok KE, Hanenberg H, Noblitt TW, Schroeder WL, Kato I, Emanuel D, Williams DA.  1998 June. High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments.  Journal of Virology 72: 4882-4892.  Abstract    Full Text

Resta R, Hooker SW, Laurent AB, Jamshedur Rahman SM, Franklin M, Knudsen TB, Nadon NL, Thompson LF. 1997 February 15. Insights into thymic purine metabolism and adenosine deaminase deficiency revealed by transgenic mice overexpressing ecto-5-nucleotidase (CD73).  The Journal of Clinical Investigations 99: 676-683.  Abstract   Full Text

Staff report, 1999 December 8. FDA says youth who died in gene therapy trial should not have received the treatment. CNN.com. Available:
<http://cnn.com/1999/HEALTH/12/08/gene.therapy.hearings/>  Accessed: April 25, 2000.

Tsuboi I, Sagawa K, Shichijo S, Yokoyama MM, Ou DW, Wiederhold MD. 1995 September. Adenosine deaminase isoenzyme levels in patients with human T-cell lymphotropic virus type 1 and human immunodeficiency virus type 1 infections. Clinical and Diagnostic Laboratory Immunology 2: 626-630.   Abstract PDF Text