ADA-deficient Mice Research Articles

Early Enzyme Replacement Therapy Improves Hearing and Immune Defects in Adenosine Deaminase Deficient-Mice.

Background: Inherited defects in adenosine deaminase (ADA) cause severe immune deficiency, which can be corrected by ADA enzyme replacement therapy (ERT). Additionally, ADA-deficient patients suffer from hearing impairment. We hypothesized that ADA-deficient (–/–) mice also exhibit hearing abnormalities and that ERT from an early age will improve the hearing and immune defects in these mice.Methods: Auditory brainstem evoked responses, organ weights, thymocytes numbers, and subpopulations, lymphocytes in peripheral blood as well as T lymphocytes in spleen were analyzed in ADA–/– and ADA-proficient littermate post-partum (pp). The cochlea was visualized by scanning electron microscopy (SEM). The effects of polyethylene glycol conjugated ADA (PEG-ADA) ERT or 40% oxygen initiated at 7 days pp on the hearing and immune abnormalities were assessed.Results: Markedly abnormal hearing thresholds responses were found in ADA–/– mice at low and medium tone frequencies. SEM demonstrated extensive damage to the cochlear hair cells of ADA–/– mice, which were splayed, short or missing, correlating with the hearing deficits. The hearing defects were not reversed when hypoxia in ADA–/– mice was corrected. Progressive immune abnormalities were detected in ADA–/– mice from 4 days pp, initially affecting the thymus followed by peripheral lymphocytes and T cells in the spleen. ERT initiated at 7 days pp significantly improved the hearing of ADA–/– mice as well as the number of thymocytes and T lymphocytes, although not all normalized.Conclusions: ADA deficiency is associated with hearing deficits and damage to cochlear hair cells. Early initiation of ERT improves the hearing and immune abnormalities.

Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

764. Dosing and Re-Administration of Intravenous Lentiviral Vector for Liver-Directed Gene Transfer in Young Rhesus Monkeys and ADA-Deficient Mice

Liver-directed gene transfer with lentiviral vectors (LV) is a treatment modality being investigated for a number of genetic diseases. To study re-administration of LV, rhesus monkeys and Adenosine deaminase (ADA) knockout mice were repeatedly dosed with vesicular stomatitis virus (VSV) pseudotyped LV. In some studies the LV were modified with polyethylene glycol (PEG) since others showed that PEG modification of lysine residues of VSV increases LV half-life in serum and reduces compliment-mediated inactivation. ADA-deficiency is a defect in purine metabolism that results in severe combined immunodeficiency due to substrate accumulation. Unlike humans, ADA-/- mice do not survive to day 21 if not treated, however, when treated with intravenous ADA LV, high ADA expression in the liver was associated with prolonged survival and immune reconstitution. Liver vector copy number (VCN) was determined by qPCR. Anti-vector IgG concentration in plasma was determined by ELISA, using VSV LV as the capture antigen and a mouse anti-VSV IgG standard. Untreated animals have 10 µg/ml of reactive IgG. Direct competitive ELISA was used to determine the PEG concentration on PEG LV. ADA-/- mice treated with 5×10e10TU/kg of ADA LV at birth did not develop anti-vector antibodies (n=12) and showed 0.3±0.23 liver VCN whereas ADA-/- mice immune reconstituted with ERT and treated with 1.5×10e10 TU/kg at 4 months (m) produced 100-fold more anti-vector IgG (n=5; 6890±1143µg/ml; p=0.01) with no difference in VCN when adjusted for dose. Similar results were found in rhesus monkeys treated with 2.0×10e9 TU/kg of LV at 1 month postnatal age (n=2; 4030±10µg/ml) compared to rhesus monkeys treated at 4 m postnatal age (n=2; 17800±300µg/ml). In immune competent ADA+/- mice treated with ADA LV at birth followed by unmodified enhanced green fluorescent protein (eGFP) LV (n=3; 0.05 VCN; 1390µg/ml) or PEG modified eGFP LV (n=3; 0.05 VCN; 1330µg/ml) at 8 m, the liver VCN and the anti-vector responses were not different. However, if mice were treated with an unmodified eGFP LV or PEG modified eGFP LV at 4 m and an unmodified ADA LV at 8 m, anti-vector IgG concentration was increased 10-fold with the unmodified eGFP LV (n=3; 114666±666µg/ml) compared to the PEG modified eGFP LV (n=3; 9733±333µg/ml); however, the second LV was inactivated and no vector was detected from mice in either group. When ADA+/- mice were treated with an unmodified ADA LV at birth and again at 5 m (booster), followed by an unmodified (n=5) or PEG modified (n=5) eGFP LV at 8 m, the third injection was associated with acute toxicity and reduced probability of survival in both groups (40%; p=0.01). In immune competent rhesus monkeys dosed with an unmodified LV carrying a non-expressed transgene at 3 m of age and followed by an unmodified LV or a PEG modified LV carrying a different non-expressed transgene at 6 m, the second vector was not detected in either group and both the unmodified LV (n=2; 15600±1,200µg/ml) and the PEG-modified LV (n=2; 15500±900µg/ml) had similar anti-vector responses. In conclusion, LV dosing at birth is associated with reduced anti-vector responses compared to dosing at ~3 m of age. Repeat LV dosing can result in inactivation of a second vector administration that is associated with higher anti-vector IgG responses and a higher probability of acute toxicity when dosed months apart. PEG modification does not appear to protect the second dose from inactivation or prevent toxicity.

442. Decreased Survival and Engraftment in Adult ADA-Deficient Mice Co-Transplanted With Uncorrected Lineage-Positive Bone Marrow Cells Combined With Gene-Corrected Lineage-Negative Bone Marrow Cells

ADA-deficiency primarily results in a severe combined immunodeficiency (SCID) but because it is a disorder of the purine salvage pathway the accumulation adenosine and deoxy-adenosine also causes skeletal, neurologic, hepatic and pulmonary abnormalities. Autologous transplantation of retroviral vector-mediated gene-corrected hematopoietic cells after non-myeloablative conditioning for the treatment of ADA-deficient SCID has been shown to be safe and efficacious, especially in very young children, with more variable results in older children. Previously, we have shown that ADA-deficient (ADA-/-) mice transplanted with gene-corrected whole marrow or ADA+/+ marrow had significantly higher engraftment of gene-corrected cells in the bone marrow, liver, and lung compared to transplantation with gene-corrected lineage negative (Lin-) bone marrow cells. The improved engraftment with whole marrow might be due the presence of specific cell populations and/or factors that could facilitate engraftment, including the additional ADA expressed from terminally differentiated cells in the niche during the engraftment period. In new experiments (n=3) we compared outcomes in ADA-/- mice transplanted with gene-corrected Lin- cells (5×10^5) alone (n=10) to these end-points in ADA-/- mice transplanted with gene-corrected Lin- cells combined with either gene-corrected lineage positive (Lin+) cells (5×10^6)(n=8), or with uncorrected Lin+ cells (5×10^6)(n= 10). Marrow cells were fractionated by immunomagnetic sorting and transduced with the lentiviral vector, EFS-ADA at 3 × 10^7 TU/ml (Lin- MOI 60, Lin+ MOI 6) for 24 hours and transplanted into adult (16 weeks) ADA-/- recipients conditioned with 500 cGy and maintained on enzyme replacement for one month after transplant. Unexpectedly, survival to day 100 was significantly reduced in ADA-/- mice transplanted with gene-corrected Lin- cells combined with uncorrected Lin+ cells (37%) compared to mice transplanted with either gene-corrected Lin- cells alone (90%) or with gene corrected Lin- cells co-transplanted with gene-corrected Lin+ (88%)(p<0.0001). Overall engraftment and VCN were measured in the thymus, spleen, bone marrow, lung and liver and neither was significantly improved with the addition of gene-corrected Lin+ cells when compared to transplant of gene-corrected Lin- cells alone. Even with so few ADA-/- mice transplanted with Lin- and uncorrected Lin+ cells surviving (n=3), overall engraftment in spleen was significantly lower (8%) when compared to ADA-/- co-transplanted with gene-corrected Lin+ cells (66%)(p<0.001). Likewise, these mice also had fewer splenic T and B cells numbers when compared to mice co-transplanted with gene-corrected Lin+ cells (p<0.05). Taken together with the very poor survival, we hypothesize the uncorrected Lin+ cells may place an extra burden systemically or within the bone marrow niche to decrease engraftment and survival.

Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity

AbstractErythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

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Objectives Although placental hypoxia has been speculated to contribute to the pathogenesis of preeclampsia (PE), the specific mechanisms linking placental hypoxia to the pathogenesis of PE are poorly understood. Adenosine (Ado) is a signaling molecule known to be induced by hypoxia and to orchestrate the cellular response to hypoxia. Our study aimed to examine the contribution of placental Ado signaling to the pathogenesis of PE. Methods We generated genetically-engineered pregnant mice with elevated placental Ado, resulting from the placenta-specific deficiency in adenosine deaminase (ADA), an enzyme that converts Ado to inosine. Additional pharmacologic and genetic approaches were taken to examine the significance of placental Ado signaling in these mice. To expand mouse studies to humans, we utilized human placenta samples and human villous explant culture. Results Pregnant mice harboring placentas with excess Ado due to the placental ADA deficiency displayed spontaneously-developed PE features including hypertension and proteinuria which appeared late in pregnancy and returned to base line postpartum, elevated production of sFlt-1, and small fetuses and placentas characterized by impaired vasculature. ADA enzyme therapy or genetically restoring ADA to the placentas of ADA-deficient mice successfully prevented all the defects in these mice by restoring normal Ado levels in the placentas. Among four Ado receptors, only the A 2 B Ado receptor (ADORA2B) was significantly increased in the mouse placentas with elevated Ado. Genetic deletion of ADORA2B prevented elevated placental Ado-induced PE features. Translational studies showed elevated Ado and increased ADORA2B in placentas of PE patients compared to normal controls. Finally, we demonstrated that ADORA2B activation directly induced FLT-1 gene expression and sFLT-1 secretion in cultured human villous explants. Conclusions Our studies revealed that elevated placental Ado coupled with ADORA2B activation is a previously unrecognized pathogenic placental signaling pathway contributing to PE and thereby highlights novel therapeutic targets. Disclosures T. Iriyama: None. K. Sun: None. N. Parchim: None. J. Lee: None. S. Blackwell: None. B. Sibai: None. L. Chan: None. T. Chan: None. M. Blackburn: None. R. Kellems: None. Y. Xia: None.

Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity

Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. DisclosuresNo relevant conflicts of interest to declare.

Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Adenosine Is A Common Factor Regulating Erythrocyte 2,3-Bisphosphate Induction In Normal Individuals At High Altitude and In Patients With Sickle Cell Disease

To function effectively in O2 uptake, transport and delivery, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)-O2 affinity by endogenous allosteric modulators. One of the best know allosteric modulators is 2,3-bisphophosphoglycerate (2,3-BPG), a metabolic byproduct of glycolysis synthesized primarily in erythrocytes for the purpose of regulating Hb-O2 affinity. Earlier studies demonstrated that erythrocyte 2,3-BPG levels are elevated at a high altitude and in patients with sickle cell disease (SCD). We have recently shown that adenosine, a molecule well known to be induced under hypoxic conditions, is significantly elevated in SCD and contributes to increased 2,3-BPG induction in SCD erythrocytes. However, whether adenosine contributes to increased erythrocyte 2,3-BPG production at high altitude is unknown. To address this question we recruited 24 individuals who normally lived at sea level, and placed them at high altitude for different time points. Similarly, blood was collected from 45 SCD patients and 50 controls (at sea level). Here we report that 1) Plasma adenosine and erythrocyte 2,3-BPG levels were significantly elevated in normal individuals at high altitude after 24 hours compared to sea level; 2) The elevations of both molecules are further enhanced at high altitude after 16 days; 3) Elevated circulating adenosine levels significantly correlated with increased erythrocyte 2,3-DPG levels in normal individuals at high altitude and in SCD patients. These results suggest that adenosine is a common factor responsible for elevated 2,3-BPG as a function in normal individuals and in SCD patients. To test this hypothesis we took pharmacologic and genetic approaches. We found that NECA, a stable adenosine analog, significantly increased 2,3-BPG production in cultured erythrocytes from normal and SCD mice and humans. Additional genetic and pharmacologic experiment showed that adenosine-induced 2,3-BPG production was mediated by ADORA2B receptor activation and downstream signaling by protein kinase A. To examine the in vivo physiological relevance of our findings we used adenosine deaminase (ADA)-deficient mice that accumulate high levels of circulating adenosine as a result of their enzyme deficiency. We found that elevated adenosine correlated to elevated erythrocyte 2,3-BPG and decreased Hb-O2 affinity. Experimental strategies to lower plasma adenosine or inhibit ADORA2B signaling in ADA-deficient mice resulted in reduced 2,3-BPG production and increased Hb-O2 affinity in erythrocytes. These studies provide in vivo evidence that elevated adenosine signaling via ADORA2B contributes to elevated of 2,3-BPG production and thereby triggers O2 release from erythrocytes. Taken together, our studies show that adenosine is a common factor regulating erythrocyte 2,3-BPG induction as a function of altitude and in patients with sickle cell disease. Our studies reveal numerous approaches to enhance oxygen release from oxyHb to physiologically or environmentally induced conditions of hypoxia. Disclosures:No relevant conflicts of interest to declare.

Adenosine Signaling in Priapism and Novel Therapies

Abstract 1006 Objective:Priapism is abnormal prolonged penile erection occurring without sexual interest. The condition is prevalent among men with sickle cell disease (SCD). Priapism is a urological emergency which needs early intervention to avoid the risks of penile fibrosis and eventual erectile dysfunction. Due to poorly understood pathogenesis of priapism no effective approaches to manage the disorder. Recent studies have revealed excess adenosine (Ado) in priapism via Ado A2B receptor (ADORA2B), suggesting novel therapeutic possibilities. Here, we aim to conduct preclinical studies to assess the efficacy and safety of Ado-based therapy in priapism. Materials and Methods:ADA-deficient mice (ADA−/−) and SCD Berkeley mice are two independent priapism animal models. We treated both mice with polyethylene glycol-modified ADA (PEG-ADA) to lower penile adenosine level or PSB1115, a selective ADORA2BR antagonist. The erectile function are measured by the changes of intracavernosal pressure (ICP) induced by cavernous nerve stimulation (CNS). Penile fibrosis is evaluated by histological studies and RT-PCR analysis of fibrotic marker gene. In vitro human microvascular endothelial cells (HMECs) were used to determine mechanism underlying ADORA2B-induced priapism. Results:Both ADA−/− and SCD mice with elevated Ado levels in penile tissue displayed priapic feature defined by prolonged and heightened erectile in response to CNS. Chronic reduction of accumulation of penile Ado levels by PEG-ADA enzyme therapy or PSB1115 corrected the priapic feature in both priapic animal models and further prevented progression of penile fibrosis. Significantly, both HIF-1α and eNOS mRNA level were elevated but reversed by PSB1115 treatment in penile tissues of ADA−/− mice and SCD mice. Finally, we provide in vitro direct evidence that PSB1115 treatment or siRNA knockdown HIF-1α in HMECs significantly reduced Ado-induced eNOS mRNA, implicating that ADORA2B-mediated HIF-1α induction contributes to elevated eNOS mRNA and underlies Ado-mediated priapism. Conclusions:PEG-ADA and PSB1115 are effective and safe to treat priapism and exacerbation of the disease by decreasing penile Ado levels or interfering its signaling. This study provides direct preclinical evidence for the novel and general utility of PEG-ADA enzyme therapy or ADORA2B antagonists for priapism and sets up a solid foundation for future clinical trials to assess the usefulness of Adobased therapeutics to treat priapism. Disclosures:No relevant conflicts of interest to declare.

Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction

AbstractGene therapy (GT) for adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada−/−). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010 ). Currently, rAAV vectors are being utilized in phase I/II clinical trials for cystic fibrosis, α-1 antitrypsin deficiency, Canavan's disease, Parkinson's disease, hemophilia, limb-girdle muscular dystrophy, arthritis, Batten's disease, and Leber's congenital amaurosis (Flotte et al., 1996 , 2004 ; Kay et al., 2000 ; Aitken et al., 2001 ; Wagner et al., 2002 ; Manno et al., 2003 ; Snyder and Francis, 2005 ; Maguire et al., 2008 ; Cideciyan et al., 2009 ). In this study, we present preclinical data to support the viability of an rAAV-based gene transfer strategy for cure of ADA-SCID. We report efficient transduction of a variety of postmitotic target tissues in vivo, subsequent human ADA (hADA) expression, and enhanced hADA secretion in tissues and blood, with increasing peripheral lymphocyte populations over time.

Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism

Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility. The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism. Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol-modified ADA (PEG-ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG-ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation. Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays. We found that lowering adenosine levels in penile tissues by PEG-ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG-ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism. Our studies have identified that PEG-ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation seen in two independent priapic animal models, and suggested its therapeutic possibility in men suffering from priapism.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

AbstractAdenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Enhanced Airway Inflammation and Remodeling in Adenosine Deaminase-Deficient Mice Lacking the A2B Adenosine Receptor

Adenosine is a signaling nucleoside that is generated in response to cellular injury and orchestrates the balance between tissue protection and the progression to pathological tissue remodeling. Adenosine deaminase (ADA)-deficient mice develop progressive airway inflammation and remodeling in association with adenosine elevations, suggesting that adenosine can promote features of chronic lung disease. Furthermore, pharmacological studies in ADA-deficient mice demonstrate that A(2B)R antagonism can attenuate features of chronic lung disease, implicating this receptor in the progression of chronic lung disease. This study examines the contribution of A(2B)R signaling in this model by generating ADA/A(2B)R double-knockout mice. Our hypothesis was that genetic removal of the A(2B)R from ADA-deficient mice would lead to diminished pulmonary inflammation and damage. Unexpectedly, ADA/A(2B)R double-knockout mice exhibited enhanced pulmonary inflammation and airway destruction. Marked loss of pulmonary barrier function and excessive airway neutrophilia are thought to contribute to the enhanced tissue damage observed. These findings support an important protective role for A(2B)R signaling during acute stages of lung disease.

Adenosine Signaling, Priapism and Novel Therapies

Priapism is defined as abnormal prolonged penile erection lasting at least for 4 hours occurring without sexual interest. Forty percent of sickle cell disease (SCD) patients display priapism. The disorder is dangerous and urgent given its association with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. Current strategies to manage the disorder are poor due to lack of fundamental understanding of the molecular mechanisms of priapism. Adenosine is a signaling nucleoside that elicits many pathophysiological effects by engaging membrane receptors. Recent evidence shows that adenosine may play an important role in priapism via adenosine receptor. To summarize the recent findings on the importance of adenosine signaling in the pathogenesis of priapism. Evidence in the literature on the association between adenosine signaling and the development of priapism. This article reviews the literature that relates to the contributory role of adenosine signaling in priapism in multiple animal models and humans. Excessive adenosine accumulation in the penis, coupled with increased A(2B)R signaling, contributes to priapism in two independent lines of mutant mice. One is adenosine deaminase (ADA)-deficient mice, the only animal displaying spontaneously prolonged penile erection, and the other is SCD transgenic mice, a well-accepted priapic animal model. Both polyethylene glycol-modified ADA (PEG-ADA) enzyme therapy and A(2B)R antagonists are capable of inhibiting potent corpus cavernosal vascular relaxation associated with priapic-like activity seen in both ADA-deficient mice and SCD transgenic mice, indicating that PEG-ADA enzyme therapy is likely to be a novel therapy for such a dangerous urological disorder. Overall, the research reviewed here raises the intriguing possibility that elevated adenosine signaling contributes to priapism in general and that this signaling pathway represents a potentially important therapeutic target for the treatment of priapism.

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion

AbstractAdenosine deaminase (ADA)–deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose–dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.

Pharmacological Blockade of A 2A Receptors Prevents Dermal Fibrosis in a Model of Elevated Tissue Adenosine

Adenosine is a potent modulator of inflammation and tissue repair. We have recently reported that activation of adenosine A(2A) receptors promotes collagen synthesis by human dermal fibroblasts and that blockade or deletion of this receptor in mice protects against bleomycin-induced dermal fibrosis, a murine model of scleroderma. Adenosine deaminase (ADA) is the principal catabolic enzyme for adenosine in vivo, and its deficiency leads to the spontaneous development of pulmonary fibrosis in mice. The aim of this study was to characterize further the contributions of endogenous adenosine and adenosine A(2A) receptors to skin fibrosis. Taking advantage of genetically modified ADA-deficient mice, we herein report a direct fibrogenic effect of adenosine on the skin, in which increased collagen deposition is accompanied by increased levels of key mediators of fibrosis, including transforming growth factor beta1, connective tissue growth factor, and interleukin-13. Pharmacological treatment of ADA-deficient mice with the A(2A) receptor antagonist ZM-241385 prevented the development of dermal fibrosis in this model of elevated tissue adenosine, by reducing dermal collagen content and expression of profibrotic cytokines and growth factors. These data confirm a fibrogenic role for adenosine in the skin and reveal A(2A) receptor antagonists as novel therapeutic agents for the modulation of dermal fibrotic disorders.

Genetic removal of the A2Aadenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice

Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A(2A) adenosine receptor (A(2A)R) functions to limit inflammation and promote tissue protection; however, the contribution of A(2A)R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A(2A)R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A(2A)R double knockout mice. Genetic removal of the A(2A)R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A(2A)R. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-alpha and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A(2A)R double knockout mice. These findings suggest that the A(2A)R plays a protective role in the ADA-deficient model of pulmonary inflammation.

Enhanced CXCL1 production and angiogenesis in adenosine‐mediated lung disease

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling molecule that has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The current study utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations in adenosine in vivo result in pulmonary angiogenesis. Results demonstrate substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. ADA replacement enzyme therapy resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the angiogenic chemokine CXCL1 (mouse functional homologue of human IL-8) were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its receptor, CXCR2, resulted in the inhibition of angiogenic activity, which suggests that CXCL1 signaling through the CXCR2 receptor mediated the observed increases in angiogenesis. Our findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis.