Normal ADA Research Articles

Gene therapy for adenosine deaminase severe combined immune deficiency-An unexpected journey of four decades.

Severe combined immune deficiency due to adenosine deaminase deficiency (ADA SCID) is an inborn error of immunity with pan-lymphopenia, due to accumulated cytotoxic adenine metabolites. ADA SCID has been treated using gene therapy with a normal human ADA gene added to autologous hematopoietic stem cells (HSC) for over 30 years. Iterative improvements in vector design, HSC processing methods, and clinical HSC transplant procedures have led nearly all ADA SCID gene therapy patients to achieve consistently beneficial immune restoration with stable engraftment of ADA gene-corrected HSC over the duration of observation (as long as 20 years). One gene therapy for ADA SCID is approved by the European Medicines Agency (EMA) in the European Union (EU) and another is being advanced to licensure in the U.S. and U.K. Despite the clear-cut benefits and safety of this curative gene and cell therapy, it remains challenging to achieve sustained availability and access, especially for rare disorders like ADA SCID.

KALA-AZAR WITHOUT SPLENOMEGALY: A RARE PRESENTATION

Visceral leishmaniasis is a major public health problem in Bangladesh, North East India, Nepal, Sudan and North East Brazil. In India, leishmaniasis is more prevalent in Bihar, Jharkhand, West Bengal and Uttar Pradesh. We present the case of a 55-year-old male farmer from Himachal Pradesh with complaints of fever for 2.5 months, appetite loss and weight loss for 1 month. On evaluation, he was found to have pancytopenia, transaminitis, and hyperbilirubinemia. Tropical fever serology and viral markers were negative. Blood and urine cultures were sterile, and ascitic fluid was acellular and high SAAG with normal ADA. Bone marrow was done due to non-responding pancytopenia which reveals intracellular amastigote form of Leishmania Donovani. A final diagnosis of Kala-azar without splenomegaly with moderate ascites was made as an absence of splenomegaly was the most striking aspect in our patient.

Ataxia-Telangiectasia, Associated with a Rare 1339C>T ATM mutation, presenting in infancy with Pneumocystis carinii pneumonia, and an elevated serum IgM. (35.11)

Abstract Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder manifested by progressive immunodeficiency, progressive neurologic dysfunction, and an elevated cancer predisposition. Characteristic symptoms are rarely encountered during infancy. We present the laboratory and clinical studies of a patient with respiratory distress at age 3 months due to pneumocystis pneumonia. Laboratory studies demonstrated an elevated serum IgM (840 mg/dL), recurrent neutropenia, lymphopenia (CD3, CD4, CD8, and CD19), diminished in vitro lymphocyte proliferation, a negative HIV PCR, and normal ADA and PNP levels. Flow cytometry demonstrated normal lymphocyte expression of CD40 and the CD40 ligand. Sequencing, after PCR-amplification of genomic DNA, demonstrated no apparent mutation consistent with type 2 hyper IgM syndrome. Based on a significantly elevated serum alpha-fetoprotein and early neurologic deficits, a diagnosis of A-T was suspected. Subsequently, her cells were found to be radiosensitive, based on a Colony Survival Assay. Western blot analysis of nuclear lysates of patient cells failed to detect ATM protein. Molecular analysis of the ATM gene identified a rare homozygous 1339C>T mutation (previously reported in a Druze A-T family: Hum Mutat 11:69–75, 1998). Notably, our A-T case (AT219LA) demonstrates early and atypical findings of an opportunistic infection and elevated serum IgM. The patient is currently thriving on IVIG and the prophylactic use of Septra.

T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates.

Adenosine deaminase-deficient severe combined immunodeficiency was the first disease investigated for gene therapy because of a postulated production or survival advantage for gene-corrected T lymphocytes, which may overcome inefficient gene transfer. Four years after three newborns with this disease were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-containing T lymphocytes has risen to 1-10%, whereas the frequencies of other hematopoietic and lymphoid cells containing the gene remain at 0.01-0.1%. Cessation of polyethylene glycol-conjugated adenosine deaminase enzyme replacement in one subject led to a decline in immune function, despite the persistence of gene-containing T lymphocytes. Thus, despite the long-term engraftment of transduced stem cells and selective accumulation of gene-containing T lymphocytes, improved gene transfer and expression will be needed to attain a therapeutic effect.

Two newly identified mutations (Thr233Ile and Leu152Met) in partially adenosine deaminase-deficient (ADA-) individuals that result in differing biochemical and metabolic phenotypes.

Deficiency of adenosine deaminase (ADA-) results in autosomal recessive immunodeficiency disease of varying severity. Partial ADA- [ADA deficiency in erythrocytes (RBCs) but substantial ADA in non-RBCs] has also been identified, primarily by population screening of healthy adults in Africa and newborns in New York State. Normal immune function and/or minimal elevations of toxic metabolites in childhood suggested that partial ADA deficiency was benign and therefore that six mutations identified in partially ADA-deficient newborns and expressing 8-80% of normal ADA in non-RBCs were not pathogenic. However, the lowest activity mutation (Arg211Cys) has now been reported in patients with adult-onset immunodeficiency. We have now molecularly and biochemically studied two additional individuals whom we found to represent opposite ends of the spectrum of partial ADA deficiency as to biochemical abnormalities and age of ascertainment. Homozygosity for a newly identified Leu152Met mutation expressing considerably less activity than the pathogenic Arg211Cys mutation was found in a currently healthy 10-year-old Afghanistani child (ascertained at birth). He had the highest accumulation of the metabolite dATP among 13 partially ADA-deficient patients studied, but considerably lower than in those with immunodeficiency. Homozygosity for a newly identified Thr233Ile mutation expressing somewhat greater ADA activity than Arg211Cys was found in a healthy young adult Kung individual, associated with very low metabolite concentrations. Biochemical findings and a family history suggestive of immunodeficiency in prior offspring support the idea that the Leu152Met mutation could result in disease in homozygous individuals challenged by severe environmental insult or in heterozygosity with a null mutation. The pathogenicity of the Thr233Ile mutation, as well as a previously described Ala215Thr mutation with relatively lower activity is less likely but will only be determined by long-term observation of individuals carrying these mutations. Although, in contrast to other partial mutations, neither of these two mutations are at CpG hot spots, the frequency of CpG mutations remains high for partial mutations but is also similarly high in ADA- immunodeficient patients (5/8 vs 12/21).

Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deaminase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene. Amendment to clinical research project, Project 90-C-195, January 10, 1992.

Significant increases in lymphocyte adenosine deaminase activity, T cell numbers and immune function have been achieved in the two children with SCID thus far treated with autologous T cells genetically-corrected by retroviral-mediated insertion of a normal ADA gene. Although the data obtained to date demonstrate that the use of ADA gene corrected peripheral T cells appears to be an effective treatment for ADA(-)SCID, it is theoretically preferable to try to develop a treatment for these children that will result in stem cell gene correction. The genetic correction of T cell progenitors with long-term immune reconstituting ability would be more desirable because repeated infusions of genetically altered cells should not be necessary and the generation of a more complete repertoire of T cell specificities might also be possible. Furthermore, the present treatment protocol involves indefinite continuation of enzyme replacement treatment with PEG-ADA. The demonstration of ADA gene expression in the progeny of transduced stem cells may simplify the decision concerning cessation of this very costly enzyme treatment (approximately $250,000/yr./patient). Recent evidence suggests that a small fraction of bone marrow or peripheral blood mononuclear cells bearing the CD34 antigen contains hematopoietic stem cells with both lymphoid and myeloid reconstituting ability. We propose in this amendment to supplement the infusion of human ADA gene-transduced autologous T cells in children with ADA(-)SCID with autologous peripheral blood CD34+ cells transduced with a second, readily distinguishable ADA vector.(ABSTRACT TRUNCATED AT 250 WORDS)

89 HEREDITARY RED BLOOD CELL ADENOSINE DEAMINASE OVERPRODUCTION: ASSOCIATION WITH INCREASED LEVELS OF STRUCTURALLY NORMAL ADA mRNA

A marked tissue-specific increase in erythrocyte adenosine deaminase activity is associated with an autosomal dominantly inherited hemolytic anemia. Preliminary studies suggested that the defect lay at the level of ADA mRNA translation in red blood cells. We have directly analyzed reticulocyte ADA mRNA from affected individuals. Sequencing of seven proband reticulocyte ADA cDNA clones did not reveal any base changes in coding or non-coding regions. RNase mapping demonstrated that the 5′- and 3′-untranslated regions of reticulocyte and B lymphoblast ADA mRNAs from affected individuals were identical to those of normal B lymphoblasts. However, RNase mapping failed to detect any comparable bands in RNA from control reticulocytes. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals compared to controls. These findings were obscured in the previous study by non-specific hybridization of the ADA cDNA probe to 18s ribosomal RNA. We conclude that the red cell-specific overexpression of ADA probably occurs at the level of transcription in erythroid precursors.

54 ADENOSINE DEAMINASE (ADA) EXPRESSED AT HIGH LEVEL IN T LYMPHOCYTES OF A PATIENT WITH ADA DEFICIENCY AND SEVERE COMBINED IMMUNE DEFICIENCY DISEASE (SCID)

Among patients with ADA deficiency, those with SCID have near complete absence of ADA, while some patients with severe T cell dysfunction but preservation of B cell function may have 1-3% of normal ADA activity in lymphoid cells. Patients with >5% of normal ADA activity in lymphoid cells have had no immune defect. We describe a patient with classical onset of SCID at 3 months, who was begun on red cell transfusions and immunoglobulin replacement therapy at 6 months of age. Despite long term survival, she remained severely lymphopenic and immune deficient. After starting PEG-ADA therapy at age 10 years her lymphocytes increased and became responsive to mitogens, and she developed in vivo B cell function. Just prior to starting PEG-ADA, unfractionated nucleated cells from her bone marrow were found to have 1000 nmol/h/mg protein). This observation was repeated with blood and marrow derived mononuclear cells and IL2-dependent T cells and T cell clones on 6 other occasions. HLA typing indicated the ADA+ T cells were the patient's own cells. T cell ADA had normal heat stability. Km, pl and was inhibited >99% by EHNA. In contrast to her T cells, 12 EBV transformed B cell clones from the patient had <0.3% of normal ADA activity. ADA+ T cells had normal T cell surface antigens and responsed in vitro to IL-2, mitogens, antigen and allogeneic cells. Important questions raised by these findings concern the molecular basis for the T cell ADA activity and the reason that these cells failed to proliferate and function more effectively in this patient in vivo. Acquisition of ADA activity by T lymphocyte precursor cells may not be sufficient to restore immune function in some ADA deficient patients. This possibility should be considered in view of proposals to treat ADA deficiency by somatic cell gene replacement.

Effects of chronic allopurinol therapy on purine metabolism in Duchenne muscular dystrophy

Adenine, adenosine, inosine, hypoxanthine, xanthosine, xanthine, guanine and guanosine blood levels in 11 Duchenne muscular dystrophy patients treated with allopurinol, 10 untreated patients and 8 healthy controls, were determined by HPLC. Serum ADA, PNP and 5'-NT were also determined. Untreated patients showed lower adenine (p less than 0.001) and higher adenosine, xanthine, ADA and PNP levels (p less than 0.01) than controls. Treated patients had lower adenine and higher xanthine levels (p less than 0.001), but higher hypoxanthine, xanthosine and guanine levels (p less than 0.001), than controls, with normal ADA and PNP. The changes observed in ADA and PNP levels suggest an involvement of these enzymes in accelerated degradation of purines in Duchenne dystrophy.

Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing.

Adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, we synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNAs showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These changes do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

ESTABLISHMENT AND CHARACTERIZATION OF ADENOSINE DEAMINASE (ADA)-DEFICIENT T CELL LINES

Congenital deficiency of the purine metabolic enzyme ADA causes severe combined immunodeficiency (SCID). The profound T lymphopenia characteristic of this disease has limited direct investigation of the cell most affected by deficiency of ADA. We have been successful in establishing both IL-2-dependent, non-transformed and HTLV-1 transformed T cell lines from eight consecutive ADA-deficient SCID patients. The lines obtained by either method are mature T cells by surface phenotype. These lines all display hypersensitivity to deoxyadenosine, characteristic of ADA deficiency, which is not reversed by deoxycytidine. Clones obtained by limiting dilution of one line show multiple unique rearrangeents of the T cell receptor beta gene suggesting clonal diversity among the circulating T cells of this patient, despite lymphopenia and SCID. Lines from 5 patients, all under three years old, have less than 1% normal ADA activity. Pharmacologic inhibitor studies suggest that this remaining activity is attributable to a non-specific aminohydrolase. In contrast, cells from two children ages 11 and 13 years are partially deficient with up to 10% normal ADA activity. An eighth SCID patient, previously found to totally lack erythrocyte ADA has approximately 50% normal ADA in a line derived from thymocytes. Analysis of the molecular basis for ADA deficiency in these lines shows that the majority have a grossly intact ADA gene, contain normal size ADA mRNA (1.6kb) and produce an ADA protein that is catalytically defective.

An approach to a selection system for adenosine-deaminase-positive (ADA+) cells and detection of rat ADA+ "revertants".

We have substituted deoxyadenosine or adenosine for hypoxanthine in the standard HAT selection system in an attempt to select for ADA-normal (ADA+) cells. ADA- human lymphoid line cells could not utilize deoxyadenosine as an alternative to hypoxanthine as a purine source (DAT) and failed to grow but were only somewhat inhibited in growth when adenosine was substituted for hypoxanthine (AAT). In contrast, ADA+ cells utilized adenosine or deoxyadenosine as efficiently as hypoxanthine as a purine source. Growth in DAT, but not in HAT, of an artificial mixture of one ADA+ human lymphoid cells in 1,000 ADA- cells resulted in enrichment of ADA+ cells to 25-86% of total cells. When we grew a rat ADA- cell line in two variations of the DAT system, we detected at least three electrophoretically different ADA+ patterns, one of which corresponded to normal rat ADA. These could represent "revertants."

Cloning of cDNA sequences of human adenosine deaminase.

Cloned cDNA sequences of human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been isolated from a cDNA library constructed in bacteriophage lambda gt10. The cDNA for the library was prepared from poly(A)+ RNA isolated from a human T-lymphoblast cell line, CCRF-CEM. The library was initially screened by differential plaque hybridization to labeled cDNA prepared from human T- and B-lymphoblast cell lines with a 21-fold difference in levels of translatable ADA mRNA. Two recombinants containing cloned cDNA sequences for ADA were identified by hybridization-selected translation. Both recombinants contained approximately 1,600 base pairs of inserted human DNA. Restriction maps of the two inserts were not identical. One contained approximately 40 base pairs of additional DNA toward the center of the cDNA. The cloned cDNA specifically hybridized to five fragments generated by HindIII digestion of human genomic DNA. It also hybridized to human lymphoblast RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast cell lines. ADA mRNA levels correlate closely with levels of ADA catalytic activity and ADA protein in cell lines containing structurally normal ADA. A leukemic T-lymphoblast line produced 6 to 9 times as much ADA protein and ADA mRNA as transformed B-lymphoblast lines. Two mutant B-lymphoblast lines from patients with hereditary ADA deficiency contained unstable ADA protein but had 3 to 4 times the normal level of ADA mRNA.

Adenosine deaminase polymorphism. Associations at clinical level suggest a role in cell functions and immune reactions.

It is well known that subjects homozygous for a rare silent allele of ADA may experience a severe combined immunodeficiency. By analogy we have investigated the possible relationship of normal ADA polymorphism with some situations, such as reproductive defects and fetomaternal interactions, in which immunological mechanisms may play an important role. A total of 572 consecutive newborns, 93 consecutive low birthweight infants, 46 couples with unexplained habitual abortion, and 24 couples with unexplained sterility were studied. The proportion of ADA 2-1 phenotype was reduced in couples with reproductive defects. In the sample of consecutive newborns the proportion of ABO incompatible babies was higher among ADA 2-1 than among ADA 1 types. ADA 2-1 phenotype was also associated with a reduction in the variability of gestational length. These associations were much more marked among male than among female babies. The proportion of ADA 2-1 was significantly lower in low birthweight infants than in the consecutively studied infants and normal adults. The present data suggest that biochemical variability resulting from the normal ADA polymorphism may be, at least in part, responsible for the variability of some immunological functions and related physiological variables and pathological conditions. They also provide evidence in favour of a selective advantage of ADA heterozygotes.

Adenosinedeaminase (ADA) polymorphism and immunological functions

It is well known that individuals homozygous for a rare silent allele of ADA may experience a severe combined immunodeficiency. By analogy we have investigated the possible relations of ADA polymorphism with some conditions (such as asthma and fetomaternal relationship) in which immunological mechanisms may play an important role.179 children with bronchial asthma,46 oouples with unexplained habitual abortion (AB),24 couples with unexplained sterility (ST),229 consecutive newborns (CN) and 93 consecutive low-birth-weight infants (LBW) were studied.The proportion of ADA2-1 phenotype was drastically reduced in asthmatic children (p<0.01) and in AB and ST couples (p<0.02).ADA2-1 phenotype was associated with a significant reduction (p<0.01) in the variability of gestational length in CN;furthermore,the proportion of ADA2-1 was significantly lower in LBW than in CN and adults (p<0.05).The present data indicate for the first time that genetic variability due to the normal ADA polymorphism may be,at least in part,responsible for the variable behaviour of immunological functions and related pathological conditions; they also provide evidence in favour of a selective advantage of ADA heterozygote. (Work supported by CBR)

Inhibition of PHA and NalO 4 mitogenesis by the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 2-deoxycoformycin (2-dCF)

Two adenosine deaminase inhibitors, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 2-deoxycoformycin (2-dCF), significantly inhibit the normal mitogenic response of dog peripheral blood mononuclear cells to phytohemagglutinin (PHA) and sodium periodate (NaIO 4) oxidation. The addition of excess PHA does not reverse the inhibitory effects of EHNA on the normal PHA response. However, it was further observed that if EHNA (10 −3, 10 −4 M) or 2-dCF (10 −3 M) were added to fresh cultures of murine P388 leukemia cells or Novikoff rat hepatoma cells, cellular proliferation, in general, was markedly inhibited. We then pretreated dog cells with 2-dCF and observed that even a brief exposure of cells to this compound was sufficient to inhibit the increased [ 3H]thymidine uptake of these cells in response to PHA or NaIO 4 treatment. In contrast, similar pretreatment of Novikoff or P388 cells did not alter the normal logarithmic growth phase. Therefore, we conclude that EHNA and 2-dCF may have an overall inhibitory effect on cellular proliferation when continuously present in cultures of dog mononuclear cells, P388 mouse leukemia cells, and Novikoff rat hepatoma cells. However, unlike continuous cell lines which grow normally after pretreatment with 2-dCF, it appears that there is a specific requirement for normal ADA function early in the initiation of DNA synthesis in dog cells which are responding to the nonimmunologic mitogenic stimulation of PHA and NaIO 4.

Abnormal spontaneous rosette formation and rosette inhibition in lung carcinoma.

Fifty-five children with CID and known ADA status were studies at a workshop held in Albany, New York. Erythrocyte ADA determinations were performed in 22 of the 55 patients, 13 of whom were ADA negative. The ADA defect appears to be transmitted as an autosomal recessive trait. Some patients with CID and ADA deficiency have characteristic radiologic abnormalities of the skeleton, which are not found in other illnesses. The thymus glands of all patients with CID and ADA deficiency who could be examined have evidence of thymic involution manifested by presence of Hassall's corpuscles and differentiated germinal epithelium; this is in contrast to "classic" thymus findings in CID with normal ADA. Adenosine deaminase probably plays an important, although as yet undefined, role in lymphocyte development and/or function. The deficiency of ADA in CID is the first enzyme defect observed in a deficiency disease of specific immunity.

Combined immunodeficiency disease associated with adenosine deaminase deficiency: Report on a Workshop Held in Albany, New York, October 1, 1973

Fifty-five children with CID and known ADA status were studies at a workshop held in Albany, New York. Erythrocyte ADA determinations were performed in 22 of the 55 patients, 13 of whom were ADA negative. The ADA defect appears to be transmitted as an autosomal recessive trait. Some patients with CID and ADA deficiency have characteristic radiologic abnormalities of the skeleton, which are not found in other illnesses. The thymus glands of all patients with CID and ADA deficiency who could be examined have evidence of thymic involution manifested by presence of Hassall's corpuscles and differentiated germinal epithelium; this is in contrast to "classic" thymus findings in CID with normal ADA. Adenosine deaminase probably plays an important, although as yet undefined, role in lymphocyte development and/or function. The deficiency of ADA in CID is the first enzyme defect observed in a deficiency disease of specific immunity.