Search

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.)

Adenosine deaminase (ADA) deficiency is an inborn error of metabolism affecting multiple systems and causing severe combined immunodeficiency. We tested intravenous administration of recombinant adeno-associated virus (AAV) 2/8-ADA vector in ADA-deficient neonate and adult mice or as part of a bimodal approach comprised of rAAV treatment at birth followed by infusion of lentiviral vector (LV)-modified lineage-depleted bone marrow cells at 8 weeks. ADA−/− mice treated with rAAV and enzyme replacement therapy (ERT) for 30 days were rescued from the lethal pulmonary insufficiency, surviving out to 180 days without further treatment. rAAV vector copy number (VCN) was highest in liver, lung, and heart and was associated with near-normal ADA activity and thymocyte development. In the bimodal approach, rAAV-mediated ADA expression supported survival during the 4 weeks before infusion of the LV-modified bone marrow cells and during the engraftment period. Conditioning prior to infusion may have resulted in the replacement of rAAV marked cells in marrow and liver, with LV VCN 100- to 1,000-fold higher in hematopoietic tissue compared with rAAV VCN, and was associated with immune cell reconstitution. In conclusion, a bimodal approach may be an alternative for patients without reliable access to ERT before receiving a stem cell transplant or gene therapy., Graphical Abstract, ADA-deficient mice were treated with rAAV8-ADA alone or as a bimodal gene therapy, with rAAV given at birth and LV-mediated ex vivo HSPC gene therapy after post-natal day 60. Treated mice survived 180 days with either approach, demonstrating rAAV-ADA may be an alternative for patients without reliable access to enzyme replacement.

Adenosine deaminase (ADA)-deficient mice and healthy rhesus monkeys were studied to determine the impact of age at treatment, vector dosage, dosing schedule, repeat administration, biodistribution, and immunogenicity after systemic delivery of lentiviral vectors (LVs). In Ada−/− mice, neonatal treatment resulted in broad vector marking across all tissues analyzed, whereas adult treatment resulted in marking restricted to the liver, spleen, and bone marrow. Intravenous administration to infant rhesus monkeys also resulted in dose-dependent marking in the liver, spleen, and bone marrow. Using an ELISA to monitor anti-vector antibody development, Ada−/− neonatal mice did not produce an antibody response, whereas Ada−/− adult mice produced a strong antibody response to vector administration. In mice and monkeys with repeat administration of LV, a strong anti-vector antibody response was shown in response to the second LV administration, which resulted in LV inactivation. Three separate doses administered to immune competent mice resulted in acute toxicity. Pegylation of the vesicular stomatitis virus G protein (VSV-G)-enveloped LVs showed a less robust anti-vector response but did not prevent the inactivation of the second LV administration. These studies identify important factors to consider related to age and timing of administration when implementing systemic delivery of LVs as a potential therapeutic agent. Keywords: lentiviral vector, gene therapy, rhesus monkeys, in vivo, immune response, ADA-deficiency, ERT, repeat administration

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)–ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Retroviral gene therapy has proved efficacious for multiple genetic diseases of the hematopoietic system, but roughly half of clinical gene therapy trial protocols using gammaretroviral vectors have reported leukemias in some of the patients treated. In dramatic contrast, 39 adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients have been treated with 4 distinct gammaretroviral vectors without oncogenic consequence. We investigated clonal dynamics and diversity in a cohort of 15 ADA-SCID children treated with gammaretroviral vectors and found clear evidence of genotoxicity, indicated by numerous common integration sites near proto-oncogenes and by increased abundance of clones with integrations near MECOM and LMO2 These clones showed stable behavior over multiple years and never expanded to the point of dominance or dysplasia. One patient developed a benign clonal dominance that could not be attributed to insertional mutagenesis and instead likely resulted from expansion of a transduced natural killer clone in response to chronic Epstein-Barr virus viremia. Clonal diversity and T-cell repertoire, measured by vector integration site sequencing and T-cell receptor β-chain rearrangement sequencing, correlated significantly with the amount of busulfan preconditioning delivered to patients and to CD34+ cell dose. These data, in combination with results of other ADA-SCID gene therapy trials, suggest that disease background may be a crucial factor in leukemogenic potential of retroviral gene therapy and underscore the importance of cytoreductive conditioning in this type of gene therapy approach.

Adenosine deaminase (ADA)-deficient mice and healthy rhesus monkeys were studied to determine the impact of age at treatment, vector dosage, dosing schedule, repeat administration, biodistribution, and immunogenicity after systemic delivery of lentiviral vectors (LVs). In Ada−/− mice, neonatal treatment resulted in broad vector marking across all tissues analyzed, whereas adult treatment resulted in marking restricted to the liver, spleen, and bone marrow. Intravenous administration to infant rhesus monkeys also resulted in dose-dependent marking in the liver, spleen, and bone marrow. Using an ELISA to monitor anti-vector antibody development, Ada−/− neonatal mice did not produce an antibody response, whereas Ada−/− adult mice produced a strong antibody response to vector administration. In mice and monkeys with repeat administration of LV, a strong anti-vector antibody response was shown in response to the second LV administration, which resulted in LV inactivation. Three separate doses administered to immune competent mice resulted in acute toxicity. Pegylation of the vesicular stomatitis virus G protein (VSV-G)-enveloped LVs showed a less robust anti-vector response but did not prevent the inactivation of the second LV administration. These studies identify important factors to consider related to age and timing of administration when implementing systemic delivery of LVs as a potential therapeutic agent.

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme of purine metabolism encoded by the Ada gene, is a cause of human severe combined immune deficiency (SCID). Numerous deleterious mutations occurring in the ADA gene have been found in patients with profound lymphopenia (T− B− NK−), thus underscoring the importance of functional purine metabolism for the development of the immune defense. While untreated ADA SCID is a fatal disorder, there are multiple life-saving therapeutic modalities to restore ADA activity and reconstitute protective immunity, including enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) with autologous gene-corrected hematopoietic stem cells (HSC). We review the pathogenic mechanisms and clinical manifestations of ADA SCID.

Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)–deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA −/− mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA −/− mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34 + cells transduced with 1–5 × 10 7 TU/ml had 1–3 vector copies/cell and expressed 1–2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.

Background: Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a rare disorder caused by ADA gene mutations, leading to lymphotoxic build-up of purine metabolites and profound immunodeficiency. Historically, enzyme replacement therapy (ERT) has been used as a bridge therapy until patients can receive an allogeneic hematopoietic stem cell transplantation (HSCT), ideally from a matched related donor (MRD) or, if none is identified, a non-matched and/or unrelated donor. We developed a self-inactivating lentiviral vector (LV), denoted EFS-ADA LV, encoding the human ADA cDNA sequence under the control of a shortened human elongation factor 1α gene promoter. A fresh or cryopreserved formulation of a drug product (OTL-101), composed of autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with EFS-ADA LV, was evaluated in 2 prospective, non-randomized Phase I/II clinical trials at 2 USA centers. We report on safety and efficacy of OTL-101 in 30 ADA-SCID pediatric gene therapy (GT) subjects treated from 2013-2017 with a median follow up (FU) of 24 months (mo; range 12-26 mo), compared to a historical cohort of 26 ADA-SCID patients treated with HSCT. Methods: UCLA Fresh Study (NCT01852071): Autologous CD34+ HSPCs were isolated from bone marrow and pre-stimulated with cytokines before transduction with EFS-ADA LV to yield OTL-101, which was infused as a fresh formulation in 20 subjects (9 male, 11 female; aged 4 mo-4.3 yrs). Single dose busulfan (4 mg/kg) was administered prior to infusion of OTL-101. Subjects were followed for 24 mo. UCLA Cryo Study (NCT02999984): 10 subjects (4 male, 6 female; aged 5-15 mo) received a cryopreserved formulation of OTL-101, which allowed for an extended shelf-life and full quality control prior to infusion. Busulfan was administered in 2 doses, the first at 3 mg/kg and the second adjusted to target a total area under the curve of 4,900 µM*min (20 ng/mL*hr). At the time of analysis, all subjects reached 12 mo FU (except 1 subject who was withdrawn from the study due to lack of engraftment); 7 subjects reached 18 mo of FU. Historical Control Group: 26 patients (aged 0.2 mo-9.8 yrs) were treated with allogeneic HSCT (MRDs n=12, non-MRDs n=14) at Great Ormond Street Hospital, UK (n=16) or Duke University Children's Hospital, USA (n=10) from 2000-2016. Results: Sustained engraftment of genetically modified HSPCs was observed in 29/30 GT subjects by 6-8 mo and persisted through FU in both studies, based on vector gene marking in granulocytes and CD3+ T cell reconstitution (Figure). Subjects who engrafted maintained long-term metabolic detoxification from deoxyadenosine nucleotides after stopping ERT approximately 1 mo post-GT. At last FU (median 24 mo; range 12-24 mo) in the GT group, overall survival (OS) was 30/30 (100%) and event-free survival (survival in the absence of ERT reinstitution or rescue allogeneic HSCT; EvFS) was 29/30 (97%). OS and EvFS were higher in the GT group at last FU compared with HSCT controls (with or without an MRD) at 2 years (Table). One of 30 OTL-101 subjects (3%) did not engraft and was restarted on ERT; the subject was withdrawn from the study at 5.9 mo and subsequently received a rescue HSCT, whereas 42% of HSCT patients required rescue HSCT, PEG-ADA ERT or died. Among the 20 OTL-101 subjects in the UCLA Fresh Study who reached 2 years FU, 18 (90%) stopped immunoglobin replacement therapy (IgRT), compared to 52% of HSCT patients. Preliminary results were observed in 5/7 (71%) OTL-101 subjects in the UCLA Cryo Study with more limited (18 mo) FU. Twelve OTL-101 subjects experienced one or more serious adverse events, most frequently infections and gastrointestinal events; only 1 of which was considered treatment-related (bacteremia due to product contamination). In the GT group, there were no events of autoimmunity with ≤24 mo FU. Due to the autologous nature of OTL-101, there was no incidence of graft vs host disease (GvHD); in contrast, 8 HSCT patients experienced GvHD events (5 acute, 3 chronic events), 1 of which resulted in death. Conclusions: Based on sustained gene correction and restoration of immune function in all subjects who engrafted, treatment of ADA-SCID with OTL-101 has a favorable benefit-risk profile. Key correlates of engraftment were consistent across the expanded cohort. Importantly, higher rates of OS and EvFS compared with HSCT (with or without an MRD) were observed. Disclosures Kohn: Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor on IP licensed from UC Regents to Orchard Therapeutics. Future royalties may occur., Research Funding; NIH: Research Funding. Shaw:Orchard Therapeutics: Consultancy, Other: Personal fees and non-financial support; NIH: Research Funding. Carbonaro-Sarracino:NIH: Other: Salary while working on project at UCLA 2013-2016, Research Funding; Orchard Therapeutics: Consultancy, Employment. De Oliveira:National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding. Terrazas:California Institute for Regenerative Medicine: Research Funding; Gene Therapy Resource Program, NHLBI/NIH: Research Funding. Hollis:Curative Therapeutics: Consultancy, Other: Personal fees. Trevisan:Orchard Therapeutics: Research Funding. Arduini:Orchard Therapeutics: Employment, Equity Ownership. Lynn:Orchard Therapeutics: Employment, Equity Ownership. Kudari:Orchard Therapeutics: Employment, Equity Ownership. Spezzi:Orchard Therapeutics: Employment, Equity Ownership. Buckley:Duke University: Research Funding. Booth:SOBI: Consultancy; GSK: Honoraria; NovImmune: Consultancy. Thrasher:Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard Therapeutics: Employment, Equity Ownership, Patents & Royalties: Lentiviral vector for gene therapy of ADA-SCID.

Adenosine 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.

A patient with adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells.

Using a mouse model of adenosine deaminase-deficient severe combined immune deficiency syndrome (ADA-deficient SCID), we have developed a noninvasive method of gene transfer for the sustained systemic expression of human ADA as enzyme replacement therapy. The method of delivery is a human immunodeficiency virus 1-based lentiviral vector given systemically by intravenous injection on day 1 to 2 of life. In this article we characterize the biodistribution of the integrated vector, the expression levels of ADA enzyme activity in various tissues, as well as the efficacy of systemic ADA expression to correct the ADA-deficient phenotype in this mouse model. The long-term expression of enzymatically active ADA achieved by this method, primarily from transduction of liver and lung, restored immunologic function and significantly extended survival. These studies illustrate the potential for sustained in vivo production of enzymatically active ADA, as an alternative to therapy by frequent injection of exogenous ADA protein.

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (

Mucopolysaccharidosis type I (MPS I) is a lysosomal glycosaminoglycan (GAG) storage disorder caused by deficiency of alpha-l-iduronidase (IDUA). In this study, we evaluated the potential to perform gene therapy for MPS I by direct in vivo injection of a lentiviral vector, using an IDUA gene knockout murine model. We compared the efficacy in newborn versus young adult MPS I mice of a single intravenous injection of the lentiviral vector. The extent of transduction was dose-dependent, with the liver receiving the highest level of vector, but other somatic organs reaching almost the same level. The phenotypic manifestations of disease were partially improved in the mice treated as young adults, but were nearly normalized at every end-point measured in the mice treated as neonates. In the neonatally treated mice, the expressed IDUA activity resulted in decreased GAG storage, prevention of skeletal abnormalities, a more normal gross appearance, and improved survival. Most strikingly, significant levels of IDUA enzyme were produced in the brain of mice treated as neonates, with transduction of neurons at high levels. The sustained expression of enzymatically active IDUA in multiple organs had a significant beneficial effect on the phenotypic abnormalities of MPS I, which may be translated to clinical gene therapy of patients with Hurler disease.

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.

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34(+) cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).

Site-specific analysis of tyrosine hydroxylase phosphorylation in rat pheochromocytoma led previously to the identification of a novel growth factor-sensitive serine/threonine protein kinase, designated proline-directed protein kinase (PDPK). In this article we describe further the activation, purification, subunit configuration, and biochemical characteristics of this cytoplasmic enzyme system. In human A431 epidermoid carcinoma cells PDPK activity was found to be stimulated by epidermal growth factor in a dose-dependent, time-dependent manner. The PDPK purified from the cytosol of mouse FM3A mammary carcinoma cells exhibited the same chromatographic behavior and biochemical properties as the tyrosine hydroxylase-associated enzyme purified originally from rat pheochromocytoma. The presence of p34cdc2 was ultimately detected in all active fractions of highly purified PDPK by Western blotting and immunoprecipitation; however, it was determined that this catalytic subunit is complexed with a 58-kDa regulatory subunit that is clearly distinct from that of the "growth-associated" M phase-specific histone H1 kinase (i.e. cyclin B). The 58 kDa regulatory subunit of PDPK was identified by direct immunoblotting as a mammalian A-type cyclin. Furthermore, the p58cyclin A subunit of PDPK was found to be phosphorylated on tyrosine residues in vivo and in vitro, the latter of which resulted in a significant increase in PDPK activity. Additional distinctions between this growth factor-sensitive PDPK (p34cdc2-p58cyclin A) and the M phase-specific histone H1 kinase (p34cdc2-p62cyclin B-p13suc1) are identified on the basis of chromatographic behavior, enzyme kinetics, and physicochemical properties. Based on these findings, it is proposed that PDPK represents a unique complex of the p34cdc2 protein kinase which is active in the cytoplasm of proliferative cells, is regulated differently from the M phase-specific histone H1 kinase by phosphorylation reactions, and is modulated selectively by growth factors.

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

ADA deficiency in humans results in a SCID phenotype, characterized by extremely low levels of T, B and NK cells, and other organ systems also may be affected (skeletal, hepatic and CNS). In mice, however, knockout of the ADA gene is lethal because ADA is required for gestation. A viable murine model of ADA- deficient SCID was developed in which the ADA-deficient embryos are rescued with an ADA mini-gene expressed only during gestation. Post-natally, these mice die from a non-infectious pulmonary insufficiency by day 19-20, which can be rescued using enzyme replacement therapy (ERT) with PEG-ADA. In these studies, newborn ADA-deficient pups (whole litters) were transplanted with normal congenic bone marrow (5.0 |[times]| 10^6 cells in 100 ul PBS via the superficial temporal vein). These non-ablated neonatal congenic BMT resulted in long-term survival (up to 360d) and multi-system improvement, including restored fertility and immune function. To characterize the extent and nature of the graft, we have analyzed these mice at 16, 60 and 180 days after neonatal BMT, compared to normal mice and to ADA-deficient mice maintained on ERT. By day 16, untreated ADA deficient pups (n=4) were profoundly lymphopenic with dramatically decreased levels of thymic T-cells, splenic T-cells and B-cells, and no lymphocyte proliferation in response to stimulation with ConA. Thymocyte and splenocyte cell population numbers and function were improved at 16, 60 and 180 days in neonatal recipients of normal congenic bone marrow, and while sub-normal in several aspects, were consistently better than in mice maintained on ERT. The levels of donor cell chimerism in recipient tissues (thymus, spleen, bone marrow, liver and lung) were similar, in the range of 0.3-3.0% (determined by Q-PCR and Southern blot analysis for the normal ADA gene allele). We obtained increases in the chimerism of donor cells in each of the cell compartments of recipients by administering low dosages of total body irradiation to the neonatal pups prior to BMT. ADA deficient pups born from dams receiving busulfan on d18 of gestation (n=7) had higher levels of chimerism than those receiving no ablation. In similarly-treated neonatal common |[gamma]|c gene knockout mice (X-linked SCID) receiving normal congenic bone marrow, we observed mostly donor-derived cells in the lymphoid compartment, in contrast to myeloid engraftment that was at low levels, similar to those seen in all lineages in the ADA gene knock-out mice. In summary, despite low engraftment, ADA-deficient neonates can be rescued with normal bone without further treatment. The ADA-deficient SCID mouse model does not show a selective proliferation of donor-derived, genetically normal T lymphoid cells after non-ablated BMT, suggesting that other mechanisms are responsible for the immune reconstitution.

The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.

Gene therapy is an experimental treatment modality under investigation for applications to HIV-1 infection. We have developed retroviral vectors carrying anti-HIV-1 genes, demonstrated that these genes cause significant suppression of HIV-1 replication in cultures of primary hematopoietic cells, and performed a clinical trial in pediatric AIDS patients. Four HIV-1-infected children and adolescents underwent bone marrow harvest from which CD34+ cells were isolated and transduced by a retroviral vector carrying an RRE decoy gene. The cells were reinfused into the subjects, without complications, showing that gene transfer in pediatric AIDS patients is safe and feasible. However, gene-containing leukocytes in the peripheral blood were seen only at a low level and only in the first months following cell infusion. To attain some degree of efficacy, it will be necessary to achieve a higher level of gene transfer and to obtain sustained gene expression. We are currently developing new gene transfer methods and vectors designed to improve the results in future trials. If it becomes possible to reach the ideal goal of producing high percentages of T lymphocytes and monocytic cells that are resistant to HIV-1 infection, gene therapy could serve as a complement to antiretroviral drug therapy and help to sustain immunologic function.

Genetic modification of hematopoietic stem cells with genes that inhibit replication of human immunodeficiency virus-1 (HIV-1) could lead to development of T lymphocytes and monocytic cells resistant to HIV-1 infection after transplantation. We performed a clinical trial to evaluate the safety and feasibility of this procedure, using bone marrow from four HIV-1–infected pediatric subjects (ages 8 to 17 years). We obtained bone marrow, isolated CD34+ cells, performed in vitro transduction with a retroviral vector carrying arev-responsive element (RRE) decoy gene, and reinfused the cells into these subjects with no evidence of adverse effects. The levels of gene-containing leukocytes in peripheral blood samples in the 1 year after gene transfer/cell infusion have been extremely low. These observations support the potential of performing gene therapy for HIV-1 using hematopoietic cells, but emphasize the need for improved gene transfer techniques.

Retroviral vectors based on the Moloney murine leukemia virus (Mo-MuLV) are among the most commonly used vectors for stable gene transfer into mammalian cells. However, expression from the transcription unit of the Mo-MuLV long terminal repeat (LTR) has often been unsatisfactory. Transcriptional suppression of retroviral vectors in vitro in embryonal carcinoma (EC) cells and in vivo in hematopoietic stem cells (HSCs) has been associated with increased levels of cytosine methylation in the vector 5' LTR. To obtain a comprehensive picture of the methylation pattern in the 5' LTR of retroviral vectors, we employed the bisulfite genomic sequencing technique, which allows detection of the methylation pattern of every CpG dinucleotide in a target sequence. We studied the 5' LTR within the Mo-MuLV-based vector, LN, and a series of multiply modified vectors, which show improved expression in vitro and in vivo. Methylation patterns of the vectors were compared in PA317 (3T3-derived) fibroblasts, which are permissive for expression from all of the vectors, and in F9 embryonal carcinoma (EC) cells, which are restrictive for expression from the parental Mo-MuLV LTR but show improved expression from the modified vectors. These analyses revealed that the levels of methylation of CpG dinucleotides were globally consistent throughout the entire LTR, including the region of transcriptional factor binding. All vectors showed no measurable methylation of CpG dinucleotides throughout the 5' LTR in the PA317 fibroblasts. The CpG dinucleotides of the standard Mo-MuLV-based vector (LN) were highly methylated in F9 EC cells (49.1%). The doubly modified vector, MD-neo, which did not show improved expression, exhibited a relatively high level of methylation (45%), similar to that found in the LN vector. In contrast, the CpG dinucleotides of the triply modified vectors, which showed improved expression in EC cells (MND-neo and MTD-neo), were much less methylated (26.2 and 23.4%, respectively). The results extend our previous findings of an inverse correlation between gene expression and methylation of cytosine residues of the LTR of retroviral vectors.

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.

Genetic deficiency of adenosine deaminase (ADA) is responsible for approximately 20% of human Severe Combined Immune Deficiency (SCID). A murine model of ADA-deficient SCID was produced by Blackburn and Kellems (U. of Texas) by ADA gene knock-out. These mice die from non-infectious pulmonary insufficiency between days 19|[ndash]|20 post-natal, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. We previously reported that neonatal mice injected with a lentiviral vector expressing hu ADA have increased survival (ASGT 7th annual meeting, June 2004). We have continued our efforts to characterize the effects of systemic ADA enzyme expression in this model of ADA-deficient SCID, using a SIN HIV-1-based lentivirus, SMPU-R-MND-ADA, pseudotyped with VSV-G and given via the temporal vein on day 2 of life. Viral supernatant was produced at high titer (1|[times]| 10^10 TU/ml). When mice received a dosage of 1.0 |[times]| 10^7 TU/kg surivial was not prolonged, but when mice received a dosage of 1.6 |[times]| 10^8 TU/kg, survival was significantly prolonged. An initial cohort of mice received PEG-ADA ERT until day 45 after neonatal injection of vector (n=4). A second cohort of mice was injected with the lentiviral vector as neonates and never given ERT (n=5). Both cohorts had survival greater than 180 days without further treatment. Mice were either sacrificed at 2 or 6 months of age. Immunophenotype analysis revealed the absence of a SCID phenotype with normal numbers of T and B lymphocytes and adequate lymphocyte proliferation in response to stimulation with ConA. Vector proviral copy number was determined by Q-PCR in peripheral blood, lung, liver, thymus, spleen and bone marrow. Proviral marking was highest in the liver (1.2+/|[minus]|0.18 copies/cell) and lung (0.12+/|[minus]|0.013 copies/cell), as has been reported for multiple bio-distribution studies of lentivirus (and AAV) given by tail vein to mice. Proviral marking in the thymus (0.0024+/|[minus]|0.0001 copies per cell), spleen (0.0037+/|[minus]|0.0001 copies/cell) and bone marrow (0.0023+/|[minus]|0.0001 copies/cell) were at least 10|[ndash]|100 fold lower than the level of marking observed in the liver and lung tissues analyzed. T-cells (CD4/8) sorted from thymus or B-cells (CD19) sorted from spleen had proviral marking similar to the marking observed in the organs from which the cells were isolated. Peripheral blood marking was very low at 2 mo (0.00007+/|[minus]|.0006 copies/cell) and undetectable by 6 mo. To determine if there was transduction of hematopoietic stem cells in surviving mice, bone marrow was transplanted into lethally irradiated, secondary recipients (n=6). There was no marking in the peripheral blood, bone marrow or colony forming units (CFUs) from bone marrow. Thus, the mice had restoration of immune function, despite very low levels of gene correction of T lymphocytes and hematopoietic cells. The findings suggest that ADA enzyme made in highly transduced organs (e.g. liver, lung) may be rescuing ADA-deficient T lymphocytes in trans; this approach of intravenous lentiviral vector delivery of the ADA gene may provide a novel form of in vivo enzyme replacement therapy.

Top of pageAbstract Wiskott Aldrich Syndrome (WAS) is a human X-linked recessive immunodeficiency characterized by thrombocytopenia, eczema, aberrant T cell activation and cytoskeletal organization. Two regulatory regions regulate the WASp gene. The proximal promoter,5' and adjacent to the initiation site, contains binding sites for several transcription factors such as Sp1, Ets, c-Myb and PU.1. The distal promoter lies 6 kb upstream from the initiation site and is followed by an alternative first exon of the WASp gene. Expression of a transferred WASp cDNA using the WASp promoter may yield more effective correction than a constitutive viral promoter. More importantly, the WASp gene enhancer/promoter may be less likely to trans-activate cellular genes in genomic sequences flanking vector integration sites, than a strong retroviral LTR with ubiquitously-active enhancers. Thus, if the WASp promoter vector shows functional equivalency to the vector using the retroviral LTR enhancer/promoter, it may represent a safer construct for clinical applications. To test the feasibility of using the endogenous WASp promoter in SIN lentiviral (SMPU) vectors, a series of constructs were developed. Transcriptional control of eGFP was directed by either the retroviral enhancer/promoter of MND, the murine PGK gene promoter, the proximal WASp promoter, the distal WASp promoter or chimeras of the distal and proximal promoters. VSV-G pseudotyped viral supernatant was produced by transfection of 293 cells. Titre for all of the WASp promoter vectors was consistently 1–2 logs lower than MND or PGK vectors. Trandsduction of cell lines was performed with equal IU/ml resulting in no more than one proviral copy per cell. In all cells the highest level of expression (MFI) was observed from the MND LTR enhancer/promoter with the murine PGK and the WASp promoters at least 10-fold less bright. However, eGFP expression driven by the proximal WASp promoter results in preferential expression (>40%) in human T cell lines, although strict tissue specificity was not observed. The distal promoter was also active, but less than the proximal promoter. The chimeric promoters, with the distal and proximal fragments together in either order, had transcripts from both promoters, but transcripts from the proximal promoter were more abundant than from the distal promoter. Interestingly, the transcripts from the distal promoter were not of the expected size with transcription appearing to start at the 5' end of the fragment. Sequence analysis revealed a potential cryptic start site in the multicloning site at the 5' end of the distal fragment, however, mutation of this potential start site did not alter the distal promoter transcript. Analysis of the actual transcript start site is in progress. Vectors containing these promoters driving WASp expression have been constructed and shown by western analysis to express WASp. Transduction of murine WASp knockout bone marrow results in WASp expression 10 fold higher from the MND enhancer/promoter than from the proximal promoter. These constructs are being tested in the murine model of WASp and in human patient cell lines.

Adenosine deaminase (ADA) deficiency is a form of severe combined immunodeficiency (SCID) that has long been considered a good candidate for gene therapy (GTx). In 2001–2002, we treated 4 ADA-SCID patients in a clinical trial evaluating the efficacy of 2 different retroviral vectors while continuing enzyme replacement with pegylated bovine ADA (PEG-ADA). No chemotherapy was used. All patients have been monitored for 6 years. No treatment-related serious adverse events occurred. A mild transient elevation in absolute lymphocyte count (ALC) was seen in 2 patients early post-treatment, however, no durable immunologic changes were observed. Low levels (0.1–0.7%) of vector-marked peripheral blood mononuclear cells (PBMCs) persist in 2 patients treated at the age of 4–5 years. All patients remain on PEG-ADA, prophylactic antibiotics and intravenous immunoglobulins. In 2004, we revised the protocol in order to facilitate engraftment and selective advantage of gene-corrected cells by withdrawing PEG-ADA and giving busulfan (75 mg/m^2) before GTx. In November 2005, a first patient was treated who developed unexpected prolonged bone marrow (BM) aplasia. Cytogenetics revealed trisomy 8 aberrations that were found to be present on a BM specimen obtained pre-GTx (Blood2007; 109:503). Our second patient enrolled in January 2007. He received 5x10^6 CD34+ cells/kg that showed 40–200 units (U) of ADA activity (normal range 58–128). Over 6 months, this patient showed a slow increase in ALC (up to 750/mcL) and lymphocyte function. PBMC ADA activity has been up to 50U. The deoxyadenosine metabolite (dAXP) level has decreased to 500/mcL were 36 and 45 days, respectively. In >200 combined days of observation, there has been only one temperature above 38 °C, which resolved with acetaminophen. These data are consistent with the positive results of GTx for ADA-SCID obtained in Milan and London and show that PEG-ADA withdrawal and reduced conditioning improve the outcome of GTx for this disease. Longer follow-up should allow us to study engraftment of cells containing vector-specific sequences and conclude if either vector contributes more to recovery of lymphoid immunity.

In humans, the genetic deficiency of adenosine deaminase (ADA) is responsible for approximately 20% of Severe Combined Immune Deficiency (SCID) and results in severe lymphopenia. The current standard is either an allogeneic bone marrow transplant from an MHC-matched donor or enzyme replacement therapy (ERT) with PEG-ADA; BMT from a donor other than a matched sibling has poor outcome. Because of a postulated selected advantage for corrected T cells, ADA-deficient SCID has been an early candidate disease for gene therapy. Recent studies by Aiuti and co-workers using oncoretroviral vectors to transduce CD34+ cells from the bone marrow of ADA-deficient SCID patients, together with cessation of ERT and moderate cytoreduction with Busulfan show clinical benefit, however, the relative roles of the cytoreduction and the cessation of PEG-ADA have yet to be elucidated. A murine model of ADA-deficient SCID was produced by Rodney Kellems and co-workers (U.Texas, Houston) by ADA gene knock-out. These mice die from non-infectious pulmonary insufficiency between days 19-20 post-natal, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. In these studies, we evaluated the role of ERT as a single variable on the ability of gene-corrected cells to repopulate 10-week old ADA-deficient mice that received a cytoablative dosage of total-body irradiation (900 cGy) prior to receiving ADA-deficient marrow transduced with the retroviral vector MND-MFG-huADA. These mice, previously maintained on ERT, were either continued on the same low dose ERT (n=8) or not (n=8). ADA -/- mice receiving ERT had 89% survival and those not receiving ERT had 75% survival after radiation and BMT. Mice were euthanized between 4-5 months post transplant and were perfused with PBS prior to tissue removal. Proviral marking was assessed by qPCR in the peripheral blood leukocytes, thymus, spleen, bone marrow, lung and liver, as well as in sorted cell populations from the thymus (CD4/8), spleen (CD19) and bone marrow (CD11b). Gene marking was seen in all of the tissues assessed with little difference between those animals receiving ERT and those that did not. Although not significant, marking was slightly higher in the hematopoietic tissues analyzed from mice receiving ERT. Proviral marking (copies/ cell) was highest in the thymus and spleen and lowest in the bone marrow. Cell populations enriched from these organs had marking similar to the organ from which they were isolated. Marking in the lung and liver was similar to that seen in the hematopoietic tissues and may be relevant due to the severe lung and hepatocellular pathology seen in ADA-deficient mice. With or without ERT, ADA- deficient mice receiving gene therapy had improved immune cellularity and function compared to ADA-deficient mice receiving mock transductions and maintained on ERT. In these studies, we have observed no selective lymphoid expansion of the gene-corrected T cells, whether ERT was given or withheld, but nevertheless there was immunologic improvement from host (ADA-deficient) cells.

Insertional oncogenesis has become the major safety concern for clinical gene transfer into human hematopoietic stem cells, because random retroviral vector integration may activate cellular proto-oncogenes and lead to the emergence of hyperproliferative clones. One potential approach to prevent adverse events while retaining the clinical benefits is to include a suicide gene in the retroviral vector carrying the therapeutic gene; if needed, vector-driven proliferation could be eliminated by drug treatment. Our lab has developed a novel model for experimental insertional oncogenesis in mice to assess the effectiveness of suicide gene therapy. Delivery of a bicistronic MoMLV-based retroviral vector encoding the murine cellular oncogene c-kitD814V to the IL-3 dependent murine leukemia cell line Ba/F3 resulted in vector-driven autonomous cell proliferation after IL-3 withdrawal from the culture medium. The vector was designed to simultaneously express a suicide gene from an internal ribosome entry site (IRES). We tested three different suicide gene candidates: the wild type Herpes simplex virus thymidine kinase (HSV-TK); sr39TK, a mutant created by semi-random mutagenesis that has been reported to have a much higher affinity for Ganciclovir (GCV); and "superTK", based on the sr39TK sequence with two silent point mutations introduced to eliminate cryptic RNA splice acceptor and donor site. We derived clonal cell lines from transduced Ba/F3 cells expressing c-kitD814V and either one of the three suicide genes. All cell lines were sensitive to GCV -induced cell death in vitro, and the Ba/F-sr39TK and Ba/F-superTK cells responded to GCV dosages approximately 10 fold lower than the Ba/F-HSV-TK cells. We also characterized GCV-sensitivity of heterogenous, non-selected transduced cell pools to account for suicide escape mutants that may develop due to partial vector loss, splicing events, gene rearrangements or epigenetic silencing. GCV treatment for 21 days of Ba/F3 cell pools transduced with HSV-TK or sr39TK resulted in emergence of GCV resistance; in contrast, proliferation was consistently inhibited by long-term GCV treatment of Ba/F3 cells transduced with superTK. The cell lines transduced to express c-kitD814V were leukemogenic in vivo when injected i.v. into immunocompetent, syngeneic Balb/c mice. Moreover, leukemogenicity was strictly dependent on vector insertion since mice challenged with non-transduced Ba/F3 cells did not develop leukemia. GCV treatment (50 mg/kg i.p.) of Balb/c on five consecutive days one week after challenge with 5,000 Ba/F-HSV-TK cells did not protect from leukemia development, and the mice succumbed to disease around day 21 post-challenge. In contrast, the same GCV treatment of mice injected with 5,000 Ba/F-sr39TK or Ba/F-superTK led to 100% survival 40 days post-challenge. Thus, in this model of vector-driven insertional oncogenesis, a suicide gene approach was effective for eliminating leukemia using modified HSV-TK variants with improved biological activity and diminished aberrant splicing.

Genetic deficiency of adenosine deaminase (ADA) is responsible for ~20% of human severe combined immune deficiency (SCID). While promising results have been obtained by Aiuti and co-workers using oncoretroviral vectors to transduce CD34+ cells from the bone marrow of ADA-deficient SCID patients, lentiviral vectors may lead to higher levels of ADA gene transfer that could extend benefits to older subjects with less transducible bone marrow or allow lower dosages of cytoreductive therapy to be used. We have constructed a SIN HIV-1-based lentiviral vector, SMPU-R-MND-ADA, which was produced at high titer (1 × 10e10 i.u./ml after 100-fold concentration) using VSV-G pseudotype and have shown it to express high levels of ADA enzyme activity in human and murine cell lines in vitro. A model of ADA-deficient SCID was produced by Rodney Kellems and co-workers (U.Texas, Houston) by ADA gene knock-out and rescue of neonatal lethality with an ADA mini-gene expressed in the placenta during gestation. These mice die from a non-infectious pulmonary disease within the first month of life, unless maintained on enzyme replacement therapy (ERT) with PEG-ADA. We have determined that transplantation of newborn ADA −/− mice with normal congenic bone marrow allows long-term survival without PEG-ADA and facilitates breeding.

Mucopolysaccharidosis type I (MPS I) is lysosomal storage disorder caused by deficiency of the enzyme, alpha-L-iduronidase (IDUA). We investigated gene therapy for MPS I by using a recombinant lentiviral (HIV-based) vector (pSMPU-MCU3-huID-IRES-eGFP) that contains the normal human IDUA cDNA as well as the enhanced green fluorescence protein (eGFP) reporter gene. We have demonstrated significant levels of IDUA gene expression by this vector in cell lines. To evaluate the effect of age on systemic gene transfer using this vector, we performed a single intravenous injection at different-ages in the IDUA gene knock-out mouse (developed by HH Li and EF Neufeld), treated as neonates (0 or 1-day old) (via facial vein, group 1, n=7), or as young-adults (8-week old) (via tail vein, group 2, n=5), with untreated age-matched control groups. We used the same weight-adjusted dosage of vector (1.65×10e5TU/kg) in neonates and young-adults, and sacrificed them at the same age (20-week old) to investigate the pathological changes, IDUA activity, and vector biodistribution in liver, spleen, kidney, heart, and brain. In the newborn group, almost no lysosomal storage was seen in all 5 organs (there was a little variance in the kidney). In contrast, in the young-adult group there was moderate accumulation in liver and spleen, and high levels were seen in heart, kidney, and brain. The IDUA activity was significantly higher (p

A herd of 8 Przewalski horses were observed on pasture in summer. Fifteen-minute focal animal samples were used to determine the time budget of the horses during the periods 00.00–04.00, 04.00–08.00, 08.00–12.00, 12.00–16.00, 16.00–20.00 and 20.00–24.00 h EDT. The behavioral states recorded were feeding (grazing and eating grain), nursing, drinking, standing, stand-resting, self-grooming, mutual grooming, locomoting, playing, and lying laterally and sternally. The average number of behavioral states occurring per hour, and the defecation, urination, aggression and vocalization rates were also determined. Overall, the horses spent 46.4 ± 5.9% of their time feeding, 1.3 ± 0.1% nursing, 0.5 ± 0.1% drinking, 20.6 ± 5.4% standing, 15.7 ± 3.2% stand-resting, 1.7 ± 0.2% self-grooming, 2.2 ± 0.7% mutual grooming, 7.4 ± 1.0% locomoting, 1.2 ± 0.3% playing, 1.2 ± 0.5% lying laterally and 4.1 ± 3.0% lying sternally. The horses averaged 45.2 ± 5.8 behavioral states per hour, and 0.2 ± 0.0 defecations, 0.3 ± 0.0 urinations, 1.5 ± 0.3 aggressions and 0.7 ± 0.1 vocalizations per hour. The horses spent the greatest amount of time foraging between 20.00 and 04.00 h, when the temperatures were lower. They spent 68.2 ± 2.2% of their time between 20.00 and 24.00 h feeding, but only 31.2 ± 2.1% of their time feeding between 08.00 and 12.00 h. Recumbent rest was most common between 00.00 and 04.00 h. As temperatures rose during the daylight hours, the horses spent more time drinking and standing, rather than grazing. Stand-resting was the most common form of rest during the day. The horses exhibited the greatest number of activities per hour from 08.00 to 20.00 h. While standing in close proximity to one another during these hours, the horses exhibited the highest number of aggressions per hour (1.9–2.4).

Pregnant and lactating pony mares were observed in two environments, stable and pasture. Twenty-six pony mares were observed on pasture for 2 weeks before and after parturition. The behavior of each mare was recorded every 30 min from 18.00 to 06.00 h. The mutually exclusive behaviors were standing (either standing alert or standing at rest with a hindlimb flexed), grazing (prehending or masticating grass), walking, lying in sternal recumbency and lying in lateral recumbency. The total time-budget for prepartum mares on pasture was 55.3 ± 4.1% grazing, 32.9 ± 3.3% standing, 6.0 ± 1.5% lying in sternal recumbency, 2.7 ± 0.7% walking and 1.4 ± 0.6% lying in lateral recumbency. Grazing and standing occurred at all times, but grazing was most common from 18.00 to 21.00 h and after 05.00 h. Lying was most common between 01.00 and 04.00. Lying in lateral recumbency occurred only after dark, in pre-partum mares. The total time-budget for post-partum mares on pasture was 68.6 ± 4.0% grazing, 22.5 ± 3.0% standing, 4.7 ± 1.0% walking, 4.2 ± 1.2% lying in sternal recumbency and 0.2 ± 0.2% lying in lateral recumbency. Lying in lateral recumbency was seen only at 18.00 h. Lying in sternal recumbency occurred between 21.00 and 04.30 h. More time was spent grazing by the post-partum mares than by the pre-partum mares. The same behaviors were recorded for stabled pony mares except that eating hay rather than grazing constituted the ingestive behavior quantified. The total time-budget for pregnant stabled ponies was 71 ± 3% standing, 15 ± 3% eating, 0.5 ± 0% lying in lateral recumbency, 0.5 ± 0.2% walking and 12.1 ± 2.3% lying in sternal recumbency. Eating decreased and standing increased during the night. Most lying was seen between 01.30 and 05.00 h. Lying in lateral recumbency occurred between 19.30 and 03.30 h. The total nocturnal time-budget of post-partum stalled ponies was 67 ± 3% eating, 19 ± 3% standing and 13.0 ± 2.3% lying in sternal recumbency. Post-partum mares were not observed to walk or to lie in lateral recumbency. The change in behavior after parturition may reflect: (1) nutritional demands of lactation; (2) maternal protective behavior; (3) response to seasonal changes in the environment.

Previous studies have suggested that experimental alterations in adrenomedullary reactivity i.e., changes in catecholamine release in response to a standard dose of acetylcholine, may be partially accounted for by changes occurring at the level of the adrenal medulla itself, independent of both the central nervous system and the innervation of the adrenal gland. The present study was designed to investigate the morphology of adrenal chromaffin cells in rats subjected to chronic hypoglycemia induced by long acting insulin, and to assess this morphology in terms of associated changes in catecholamine content and release. Surgically isolated, perfused adrenal gland preparations were utilized to characterize the functional release of catecholamines from the adrenal medulla. Pretreatment with long acting insulin resulted in a selective depletion of epinephrine stores and acetylcholine-mediated epinephrine release, but did not appear to significantly affect either the levels or the release of norepinephrine. The biochemical effects of long acting insulin persisted for several days after termination of the treatment, exhibiting a gradual recovery over a period of approximately 5 days. Electron microscopic examination of the adrenal chromaffin cells revealed a progressive degranulation and vacuolization of numerous chromaffin cells followed by a compensatory biosynthetic response and a gradual recovery toward the morphology of control cells. The functional release of catecholamines from adrenal chromaffin cells was further examined in preparations of perfused adrenal slices. Acetylcholine-mediated catecholamine release was significantly decreased in slices of adrenal glands prepared from insulin treated rats when compared with that of control animals. Taken together, our findings 1) support the hypothesis that altered adrenal reactivity is manifested largely at the level of the adrenal chromaffin cells, and 2) indicate that the chromaffin cells of the adrenal medulla represent a useful model of adrenergic cell plasticity.