ADA-deficient Patients Research Articles

New models for the study of adenosine deaminase deficiency.

Mutations in the structural gene for adenosine deaminase (ADA) which lead to a loss of enzymatic activity cause severe combined immunodeficiency (SCID) in humans. Although this disease was discovered approximately 25 years ago1 and there have been many experiments to elucidate the biochemical and immunological mechanism by which ADA deficiency leads to SCID (reviewed in 2), it is fair to say that this issue is still unsettled. For the purpose of this discussion, my remarks will be limited to the consequences of ADA deficiency on T cells, since T cells are more severely and uniformly affected in ADA-deficient patients and T cells are the focus of the next two presentations.

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).

Granulocyte colony-stimulating factor recruitment of CD34+ progenitors to peripheral blood: impaired mobilization in chronic granulomatous disease and adenosine deaminase--deficient severe combined immunodeficiency disease patients

Peripheral blood (PB) CD34+ cells mobilized by granulocyte colony- stimulating factor (G-CSF) administration are potentially useful for transplantation and as a target of gene transfer for therapy of hematopoietic disorders. Efficient harvest and planning for clinical use of PB CD34+ cells ideally requires foreknowledge of the expected mobilization kinetics and yield. We developed a sensitive flow cytometric assay for accurately enumerating CD34+ cells throughout the range seen at baseline to peak mobilization. We used this assay to assess the kinetics of G-CSF-mediated mobilization of CD34+ cells to PB in normal volunteers and in patients with chronic granulomatous disease (CGD) or adenosine deaminase (ADA)-deficient severe combined immunodeficiency disease (SCID). Two dose levels of G-CSF were examined (5 and 10 micrograms/kg/d for 7 days). Both doses were well tolerated. For normal subjects and patients an increase in PB CD34+ cells was first detected only preceding the third dose of G-CSF (day 3), peaked transiently on day 5 or 6, and then decreased thereafter despite additional doses of G-CSF. With 32 normal volunteers mean peak CD34+ cell counts were 57 and 76 cells/mm2 of blood (5 and 10 micrograms doses, respectively), whereas for 18 CGD patients the mean peaks were 31 and 40 cells/mm2 of blood. For 2 ADA-deficient SCID patients studied at a G-CSF dose of 5 micrograms/kg/d, the average peak was 16 cells/mm2 of blood. For both of these patient groups mobilization of CD34+ cells to PB was impaired compared with similarly treated normal subjects (P < .05). By contrast to the kinetics of the CD34+ cell mobilization, the absolute neutrophil count (ANC) increased markedly by 6 hours after the first dose of G-CSF and then increased steadily through day 8. At days 5 and 6 (peak mobilization of CD34+ cells) the mean ANC of CGD and ADA patients was only slightly lower ( < or = 15%) than that seen with normal subjects, whereas the difference in CD34+ cell mobilization was > 48%. Thus, ANC is not a reliable surrogate to predict peak PB CD34+ cell counts and direct enumeration of PB CD34+ counts should be undertaken in decisions regarding timing and duration of apheresis to harvest a specific number of these cells. Finally, unexpected, but significant differences in the PB CD34+ cell mobilization between normal subjects and patients with inherited disorders can occur and underscores the importance of establishing the expected mobilization of PB CD34+ cells in the planning of treatment approaches using these cells.

Granulocyte Colony-Stimulating Factor Recruitment of CD34+ Progenitors to Peripheral Blood: Impaired Mobilization in Chronic Granulomatous Disease and Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Disease Patients

Peripheral blood (PB) CD34+ cells mobilized by granulocyte colony-stimulating factor (G-CSF) administration are potentially useful for transplantation and as a target of gene transfer for therapy of hematopoietic disorders. Efficient harvest and planning for clinical use of PB CD34+ cells ideally requires foreknowledge of the expected mobilization kinetics and yield. We developed a sensitive flow cytometric assay for accurately enumerating CD34+ cells throughout the range seen at baseline to peak mobilization. We used this assay to assess the kinetics of G-CSF-mediated mobilization of CD34+ cells to PB in normal volunteers and in patients with chronic granulomatous disease (CGD) or adenosine deaminase (ADA)-deficient severe combined immunodeficiency disease (SCID). Two dose levels of G-CSF were examined (5 and 10 micrograms/kg/d for 7 days). Both doses were well tolerated. For normal subjects and patients an increase in PB CD34+ cells was first detected only preceding the third dose of G-CSF (day 3), peaked transiently on day 5 or 6, and then decreased thereafter despite additional doses of G-CSF. With 32 normal volunteers mean peak CD34+ cell counts were 57 and 76 cells/mm2 of blood (5 and 10 micrograms doses, respectively), whereas for 18 CGD patients the mean peaks were 31 and 40 cells/mm2 of blood. For 2 ADA-deficient SCID patients studied at a G-CSF dose of 5 micrograms/kg/d, the average peak was 16 cells/mm2 of blood. For both of these patient groups mobilization of CD34+ cells to PB was impaired compared with similarly treated normal subjects (P < .05). By contrast to the kinetics of the CD34+ cell mobilization, the absolute neutrophil count (ANC) increased markedly by 6 hours after the first dose of G-CSF and then increased steadily through day 8. At days 5 and 6 (peak mobilization of CD34+ cells) the mean ANC of CGD and ADA patients was only slightly lower ( < or = 15%) than that seen with normal subjects, whereas the difference in CD34+ cell mobilization was > 48%. Thus, ANC is not a reliable surrogate to predict peak PB CD34+ cell counts and direct enumeration of PB CD34+ counts should be undertaken in decisions regarding timing and duration of apheresis to harvest a specific number of these cells. Finally, unexpected, but significant differences in the PB CD34+ cell mobilization between normal subjects and patients with inherited disorders can occur and underscores the importance of establishing the expected mobilization of PB CD34+ cells in the planning of treatment approaches using these cells.

Transfer of the ADA Gene into Bone Marrow Cells and Peripheral Blood Lymphocytes for the Treatment of Patients Affected by ADA-Deficient SCID

Severe combined immunodeficiency (SCID) caused by deficiency of the enzyme adenosine deaminase (ADA) is the first genetic disorder considered for human somatic cell gene therapy. ADA-SCID patients can be cured by HLA-matched sibling donor bone marrow transplantation. Alternative transplantation strategies as well as enzyme replacement are being tested in those patients who do not have a suitable matched sibling donor. Some ADA-SCID patients may not be candidates for cytoablation due to infectious damage to the lung or liver, or may have a milder phenotype that does not justify the risks associated with haploidentical bone marrow transplantation. Replacement therapy with PEG-ADA has resulted in improvement in growth, a variable increase in the number of peripheral blood lymphocytes, and a decrease in the incidence of severe infections. Another approach to the treatment of severe genetic diseases is now represented by somatic cell gene therapy. We and others have conducted experiments in vitro and in vivo that have documented that T-lymphocytes are suitable vehicles for gene transfer. Although the pluripotent stem cell remains the ideal target cell for somatic cell gene therapy of disorders of the hematopoietic system, the use of T-lymphocytes as gene therapy vehicles is specifically indicated for ADA-deficient patients where they represent the affected cells. Furthermore, the selective engraftment of T-cells only, following bone marrow transplantation, has resulted in reconstitution of cellular and humoral immunity. A model for the functional analysis in vivo of the human immune system has been utilized for the preclinical evaluation of this approach.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody responses to bacteriophage phi X174 in patients with adenosine deaminase deficiency

Adenosine deaminase (ADA) deficiency and its biochemical consequences cause severe combined immunodeficiency (SCID). Treatment strategies, designed to correct the biochemical abnormalities, include transplantation of matched bone marrow or haploidentical bone marrow stem cells, repeated partial exchange transfusions with frozen irradiated human red blood cells (RBC), or weekly injection of polyethylene glycol-modified bovine ADA (PEG-ADA). To evaluate the effect of these therapeutic options, we studied in vitro T-cell function and in vivo antibody responses to the T-cell-dependent neoantigen, bacteriophage phi X174, in 10 children with ADA-deficient SCID. In untreated patients, T-cell function was severely depressed, and only minute amounts of antibacteriophage antibody were produced. Transplantation of bone marrow from a matched sibling (one patient) or a phenotypically matched parent (one patient) resulted in a stable graft, normal T-cell function, and substantial but subnormal antibody titers to bacteriophage, with reduced memory and impaired switch from IgM to IgG. Patients receiving T-cell-depleted haploidentical bone marrow stem cells had markedly depressed antibody responses for as long as 3 years posttransplantation, despite rapidly improving T-cell function that became normal in two of four patients. Two methods of enzyme replacement were explored. During treatment with human RBC transfusions, antibody responses to bacteriophage were as severely depressed as in untreated ADA-deficient patients. Treatment with weekly injections of PEG-ADA resulted in normalization of T-cell numbers in all four patients, normal or near-normal T-cell function in two, and mildly but variably improved T-cell function in the other two patients. Quantitatively and qualitatively normal antibody responses to bacteriophage were observed in three of four patients. Assessment of antibody responses to immunization with bacteriophage phi X174 is a useful method to monitor humoral immune function in treated ADA-deficient patients and can be used to estimate when intravenous immunoglobulin (IVIG) prophylaxis may be safely discontinued.

Antibody Responses to Bacteriophage ϕX174 in Patients With Adenosine Deaminase Deficiency

Abstract Adenosine deaminase (ADA) deficiency and its biochemical consequences cause severe combined immunodeficiency (SCID). Treatment strategies, designed to correct the biochemical abnormalities, include transplantation of matched bone marrow or haploidentical bone marrow stem cells, repeated partial exchange transfusions with frozen irradiated human red blood cells (RBC), or weekly injection of polyethylene glycol-modified bovine ADA (PEG-ADA). To evaluate the effect of these therapeutic options, we studied in vitro T-cell function and in vivo antibody responses to the T-cell-dependent neoantigen, bacteriophage phi X174, in 10 children with ADA-deficient SCID. In untreated patients, T-cell function was severely depressed, and only minute amounts of antibacteriophage antibody were produced. Transplantation of bone marrow from a matched sibling (one patient) or a phenotypically matched parent (one patient) resulted in a stable graft, normal T-cell function, and substantial but subnormal antibody titers to bacteriophage, with reduced memory and impaired switch from IgM to IgG. Patients receiving T-cell-depleted haploidentical bone marrow stem cells had markedly depressed antibody responses for as long as 3 years posttransplantation, despite rapidly improving T-cell function that became normal in two of four patients. Two methods of enzyme replacement were explored. During treatment with human RBC transfusions, antibody responses to bacteriophage were as severely depressed as in untreated ADA-deficient patients. Treatment with weekly injections of PEG-ADA resulted in normalization of T-cell numbers in all four patients, normal or near-normal T-cell function in two, and mildly but variably improved T-cell function in the other two patients. Quantitatively and qualitatively normal antibody responses to bacteriophage were observed in three of four patients. Assessment of antibody responses to immunization with bacteriophage phi X174 is a useful method to monitor humoral immune function in treated ADA- deficient patients and can be used to estimate when intravenous immunoglobulin (IVIG) prophylaxis may be safely discontinued.

Genetic correction of cultured T cells from an adenosine deaminase‐deficient patient: Characteristics of non‐transduced and transduced T cells

T lymphocytes derived from peripheral blood of a patient with adenosine deaminase (ADA) deficiency were expanded in vitro. The human ADA (hADA) gene was introduced into these replicating ADA- T cells with the use of an amphotropic recombinant retrovirus carrying the hADA gene. Subsequently, infected T cells were selected on the basis of their ADA expression, by exposure to a combination of the toxic agent xylofuranosyl-adenine and the specific ADA inhibitor 2'-deoxycoformycin. CD4+ and CD8+ T cells could be infected and selected with equal efficiencies. The genetically modified T cells were shown to contain intact copies of the provirus and to express normal levels of hADA. As expected, uninfected T cells from the ADA-deficient patient displayed an increased sensitivity to 2'-deoxyadenosine. Following genetic modification, however, this sensitivity was restored to normal levels in both CD4+ and CD8+ T cells. The introduction of the hADA gene into the genome of the in vitro expanded T cells did not alter their phenotype, proliferative capacity or cytotoxic potential. These characteristics were identical to those of T cells derived from healthy individuals. These findings are of critical importance for the clinical application of hADA gene-transducted T cells.

Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells.

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.

Identification of a point mutation resulting in a heat-labile adenosine deaminase (ADA) in two unrelated children with partial ADA deficiency.

We have determined the mutation in a child with partial adenosine deaminase (ADA) deficiency who is phenotypically homozygous for a mutant ADA gene encoding a heat-labile enzyme (Am. J. Hum. Genet. 38: 13-25). Sequencing of cDNA demonstrated a C to A transversion that results in the replacement of a proline by a glutamine residue at codon 297. As this mutation generated a new recognition site in exon 10 of genomic DNA for the enzyme Alu I, Southern blot analysis was used to establish that this child was indeed homozygous for the mutation. The abnormal restriction fragment generated by this mutation was also found in a second partially ADA-deficient patient who phenotypically is a genetic compound and also expresses a heat-labile ADA (in addition to a more acidic than normal ADA) (Am. J. Hum. Genet. 38: 13-25). Sequencing of cDNA clones from the second patient established the identical codon 297 mutation. Transfection of the mutant cDNA into heterologous cells resulted in expression of a heat-labile ADA of normal electrophoretic mobility and isoelectric point, properties exhibited by the ADA in the patients' cells.

Rejection of bone marrow transplant and resistance of alloantigen reactive cells to in vivo deoxyadenosine in adenosine deaminase deficiency

Severe combined immunodeficiency disease (SCID) in patients with adenosine deaminase (ADA) deficiency is thought to result from increased levels of purine metabolites. We attempted to immunosuppress a patient with ADA deficiency and SCID using a continuous infusion of deoxyadenosine to obtain engraftment of a T cell-depleted haplocompatible parental bone marrow graft. Before administering the drug in vivo, we investigated hematopoietic colony formation in two children with ADA deficiency (including the potential recipient), the obligate heterozygote donor (father), and normal controls using deoxyadenosine and erythro-9-(2-hydroxy-3-nanyl)adenosine (EHNA), an inhibitor of ADA. Deoxyadenosine alone in concentrations as high as 100 μ M had no significant affect on erythroid (BFU-E) or myeloid (CFU-c) colony formation. However, in the presence of EHNA there was a significant reduction in BFU-E and CFU-c growth in all subjects and controls. Increasing doses of deoxyadenosine were given to one patient with ADA deficiency and SCID as a continuous 24-hr intravenous infusion. We found that there was a linear relationship between the dose administered and the plasma level; however, doses >100 mg/day were required to increase erythrocyte dATP levels. We were able to raise intracellular dATP levels to more than three times baseline with doses of deoxyadenosine of 200 mg/day. However, there were no significant effects on the absolute lymphocyte counts or the lymphocyte responses to mitogen or alloantigen, and the haploidentical marrow failed to engraft. Our results suggest that the bone marrow of ADA-deficient patients is normal with respect to standard colony formation, that inhibitors of ADA do not adequately model the deficient state, and that the immunodeficiency in ADA deficiency is not proportionately related to either the deoxyadenosine or dATP levels, both of which were significantly elevated at the time of transplantation.

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.

Reconstitution of T- and B-cell function after T-lymphocyte-depleted haploidentical bone marrow transplantation in severe combined immunodeficiency due to adenosine deaminase deficiency

A 4-month-old male received a T-lymphocyte-depleted haploidentical bone marrow transplant (BMT) for correction of severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Although previous haploidentical bone marrow transplants have been attempted in ADA-deficient SCID, complete reconstitution of both B-lymphocyte and T-lymphocyte function has not been obtained after a single transplant. In this patient, however, rapid, complete, and persistent engraftment occurred. Potential reasons for this successful reconstitution include the use of ablation by chemotherapy (busulfan, cyclophosphamide, and cytosine arabinoside), the in vitro technique of using monoclonal antibody (CT-2) and complement to deplete the donor cells of T lymphocytes, and the relative good health of the patient prior to the transplant. Further trials using this method of haploidentical BMT may prove it to be a successful method of immunologic reconstitution in ADA-deficient SCID patients for whom an HLA-identical marrow is not available.

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.

Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

RESTORATION OF IN VITRO FUNCTION OF ADENOSINE 41 DEAMINASE (ADA) DEFICIENT LYMPHOCYTES BY INTER-LEUKIN-2 (IL-2): 41

We evaluated the effects of recombinant DNA IL-2, a T-cell lymphokine which is essential for normal immune function on the responses to mitogen and alloantigen of peripheral blood mononuclear cells (PBMC) from 3 ADA deficient patients. There was significant enhancement by IL-2 of mitogenic responses to phytohemagglutinin (PHA), pokeweed mitogen (PWM) and alloantigen (MLR) in all 3 patients. Patients with ADA positive severe combined immunodeficiency disease showed no responses to IL-2:To determine the responsive cell population we measured T3, T4 and T8 expression in PBMC exposed to PHA ± IL-2. Normal PBMC respond with increased T3 cells but no change in T4 or T8 expression. The ADA deficient PBMC incubated with an ADA inhibitor, deoxyadenosine and PHA cannot be salvaged by IL-2. These results demonstrate a unique feature of the immunodeficiency in ADA deficiency and provide further evidence that purine metabolism is distinct in lymphocyte subsets. Also, the standard in vitro cell model of ADA deficiency may be significantly limited in its use in understanding the pathogenesis of this disease.

962 RESTORATION OF IN VITRO FUNCTION OF ADENOSINE DEAMINASE (ADA) DEFICIENT LYMPHOCYTES BY INTER-LEUKIN-2 (IL-2)

We evaluated the effects of recombinant DNA IL-2, a T-cell lymphoklne which is essential for normal immune function on the responses to mitogen and alloantigen of peripheral blood mononuclear cells (PBMC) from 3 ADA deficient patients. There was significant enhancement by IL-2 of mitogenic responses to phytohemagglutinin (PHA), pokeweed mitogen (PWM) and alloantigen (MLR) in all 3 patients. Patients with ADA positive severe combined immunodeficiency disease showed no responses to IL-2:To determine the responsive cell population we measured T3, T4 and T8 expression in PBMC exposed to PHA +± IL-2. Normal PBMC respond with increased T3 cells but no change in T4 or T8 expression. The ADA deficient PBMC response is almost entirely due to T3/T4 cells. Normal PBMC incubated with an ADA inhibitor, deoxyadenosine and PHA cannot be salvaged by IL-2. These results demonstrate a unique feature of the immunodeficiency in ADA deficiency and provide further evidence that purine metabolism is distinct in lymphocyte subsets. Also, the standard in vitro cell model of ADA deficiency may be significantly limited in its use in understanding the pathogenesis of this disease.

Bone marrow transplantation only partially restores purine metabolites to normal in adenosine deaminase-deficient patients.

To delineate the extent to which bone marrow transplantation provides "enzyme replacement therapy", we have determined metabolite concentrations in two patients with adenosine deaminase (ADA) deficiency treated with bone marrow transplants and rendered immunologically normal. 10 yr after engraftment of lymphoid cells, erythrocyte deoxy ATP was markedly decreased compared to the marked elevations of deoxy ATP observed in untreated patients, but was still significantly elevated (62 and 90 vs. normal of 6.0 +/- 6.0 nmol/ml packed erythrocytes). Similarly, deoxyadenosine and adenosine excretion were both markedly diminished compared to that of untreated patients but deoxyadenosine excretion was still clearly increased (20.1 and 38.6 vs. normal of less than 0.2 nmol/mg creatinine) while adenosine excretion was in the upper range of normal (7.0 and 8.1 vs. normal of 5.6 +/- 3.6 nmol/mg creatinine). Mononuclear cell deoxy ATP content was also elevated compared to normal (5.25 and 14.4 vs. 1.2 +/- 0.3). Separated mononuclear cells of bone marrow transplanted patients contain both donor lymphocytes and recipient monocytes. When mononuclear cells were depleted of the cells enriched for donor lymphocytes (i.e. monocyte depleted) was lower than that of the mixed mononuclear cells (2.2 vs. 5.26). Surprisingly, plasma adenosine was as high as in untreated ADA-deficient patients (3.2 and 1.5 vs. untreated of 0.3-3 microM). Consistent with the elevated plasma adenosine and urinary deoxyadenosine, erythrocyte S-adenosyl homocysteine hydrolase activity was diminished (0.88 and 1.02 vs. normal of 5.64 +/- 0.25). Thus, bone marrow transplantation of ADA-deficient patients not only provides lymphoid stem cells, but also partially, albeit incompletely, clears abnormally increased metabolites from nonlymphoid body compartments.

Inhibition of Methylation by Adenosine in Adenosine Deaminase-Inhibited, Phytohemagglutinin-Stimulated Human Lymphocytes

Abstract Adenosine toxicity was studied in phytohemagglutinin-stimulated, normal human, peripheral lymphocytes in order to determine whether inhibition of S-adenosylmethionine (AdoMet)-mediated transmethylation reactions might contribute to the immune defect associated with heritable deficiency of the enzyme adenosine deaminase (ADA). Cells were treated with 20 µM erythro-9-(2-hydroxy-3-nonyl)adenine to inhibit ADA and with 20 µM uridine to prevent the pyrimidine starvation known to be caused by adenosine in this system. The further addition of 25 to 100 µM adenosine inhibited the uptake of radiolabeled thymidine and leucine into acid precipitable material, and also increased intracellular levels of S-adenosylhomocysteine (AdoHcy) from 3- to 8-fold. The formation in these experiments of AdoHcy, a potent inhibitor of AdoMet-mediated transmethylation reactions, presumably occurs through the action of S-adenosylhomocysteine hydrolase. Although the physiologic function of this enzyme is to hydrolyze AdoHcy, the reversibility of the reaction and an equilibrium constant favoring synthesis allow net synthesis of AdoHcy under conditions of adenosine excess. Inclusion of 100 to 200 µM L-homocysteine thiolactone in our experiments enhanced AdoHcy accumulation and also potentiated the inhibitory effects of adenosine on radiolabeled thymidine and leucine uptake. Cytotoxic concentrations of adenosine also caused significant inhibition of in vivo DNA methylation, a process known to be AdoMet-mediated, and a good correlation between this effect and AdoHcy levels was noted. These findings indicate that adenosine toxicity in cultured human lymphocytes is at least in part due to AdoHcy accumulation and resultant inhibition of essential AdoMet-mediated methylation reactions. The relevance of these findings to the pathogenesis of the immune defect in ADA-deficient patients is not established but deserves consideration.