Christine Kinnon

The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases.

X-linked agammaglobulinaemia (XLA) is a human immunodeficiency caused by failure of pre-B cells in the bone marrow to develop into circulating mature B cells. A novel gene has been isolated which maps to the XLA locus, is expressed in B cells, and shows mutations in families with the disorder. The gene is a member of the src family of proto-oncogenes which encode protein-tyrosine kinases. This is, to our knowledge, the first evidence that mutations in a src-related gene are involved in human genetic disease.

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector

BACKGROUND X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector. METHODS Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery. FINDINGS All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes. INTERPRETATION Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity.

High-Level Transduction and Gene Expression in Hematopoietic Repopulating Cells Using a Human Imunodeficiency Virus Type 1-Based Lentiviral Vector Containing an Internal Spleen Focus Forming Virus Promoter

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (≤24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high leve...

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency–X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Chemotaxis of macrophages is abolished in the Wiskott-Aldrich syndrome

Wiskott‐Aldrich syndrome (WAS) is a rare disease characterized by microthrombocytopenia, eczema and immune deficiency. In this study a direct‐viewing chemotaxis chamber was used to analyse chemotactic responses of WAS neutrophils and macrophages in stable linear concentration gradients. In five patients with classic WAS, chemotaxis of macrophages but not of neutrophils was found to be abolished, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells. This supports the existence of an essential functional link, previously suggested by biochemical studies, between Cdc42, WAS protein (WASp) and the actin cytoskeleton in primary human macrophages. Moreover, these data suggest that Cdc42‐WASp‐mediated filopodial extension is a requirement for chemotaxis but not for chemokinesis in these cells. Abnormal directional cell motility of macrophages and related antigen‐presenting cells may play a significant part in the immune deficiency and eczema of WAS.

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Hematopoietic Stem Cell Gene Therapy for Adenosine Deaminase–Deficient Severe Combined Immunodeficiency Leads to Long-Term Immunological Recovery and Metabolic Correction

Gene therapy can restore immune and metabolic function in patients with adenosine deaminase immunodeficiency. Out of the Bubble As part of a normal day, most people will flush a toilet, open a door, or drink from a water fountain without even thinking about it—or about the lurking pathogens poised to infect us. We are afforded this luxury because of our immune system, which responds rapidly and specifically to just about anything thrown at it. Yet, for people with severe combined immunodeficiency (SCID), who carry a mutation that thwarts adaptive immunity, everyday activities can be deadly. Like the famous “bubble boy,” some people with SCID choose to live in a germ-free environment. Yet, matched hematopoietic stem cell (HSC) transplantation, which can replace the patient’s ailing immune system with functional cells from a related donor, can offer these patients a normal life. Sometimes, however, donor relatives aren’t available. Now, two new studies provide clinical support for treatment options that may allow SCID patients without matched donors to live relatively normal lives as well. One such treatment option is gene therapy. Removing HSCs from SCID patients, repairing the underlying genetic defect in these cells, and returning the repaired cells to the original host can replace the faulty immune system in SCID patients without the graft rejection or graft-versus-host disease that follows transplantation of cells from unrelated donors. Gaspar et al. do just that for two types of SCID: X-linked SCID (SCID-X1) and adenosine deaminase–deficient SCID (ADA-SCID). The authors repaired the underlying genetic defect in 10 of 10 patients with SCID-X1 and in 4 of 6 patients with ADA-SCID, resulting in the development of a functional polyclonal T cell repertoire that persisted for at least 9 years after therapy. The procedure produced minimal side effects and permitted all patients to attend typical schools. One patient in the SCID-X1 cohort developed a blood cancer, acute lymphoblastic leukemia (ALL), a complication observed in previous SCID-X1 gene therapy studies, but this patient is currently in remission. No cases of ALL developed in the ADA-SCID cohort. The promising results of these and similar studies, albeit with an increased risk of ALL in SCID-X1 patients, support the development of new safer and more efficient vectors for this and other kinds of gene therapy. Long-term follow-up of patient participants in early gene-therapy trials such as the ones described here is critical for scientists to decipher the parameters of success and failure for gene therapy in general—and for SCID-specific treatments to bubble over into the clinic. Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID) with profound depletion of T, B, and natural killer cell lineages. Human leukocyte antigen–matched allogeneic hematopoietic stem cell transplantation (HSCT) offers a successful treatment option. However, individuals who lack a matched donor must receive mismatched transplants, which are associated with considerable morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive hematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy before infusion of gene-modified cells. All patients survived, with a median follow-up of 43 months (range, 24 to 84 months). Four of the six patients recovered immune function as a result of engraftment of gene-corrected cells. In two patients, treatment failed because of disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent cytomegalovirus infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.

Long-Term Persistence of a Polyclonal T Cell Repertoire After Gene Therapy for X-Linked Severe Combined Immunodeficiency

Gene therapy results in long-term persistence of T cells in immunodeficient patients. Out of the Bubble As part of a normal day, most people will flush a toilet, open a door, or drink from a water fountain without even thinking about it—or about the lurking pathogens poised to infect us. We are afforded this luxury because of our immune system, which responds rapidly and specifically to just about anything thrown at it. Yet, for people with severe combined immunodeficiency (SCID), who carry a mutation that thwarts adaptive immunity, everyday activities can be deadly. Like the famous “bubble boy,” some people with SCID choose to live in a germ-free environment. Yet, matched hematopoietic stem cell (HSC) transplantation, which can replace the patient’s ailing immune system with functional cells from a related donor, can offer these patients a normal life. Sometimes, however, donor relatives aren’t available. Now, two new studies provide clinical support for treatment options that may allow SCID patients without matched donors to live relatively normal lives as well. One such treatment option is gene therapy. Removing HSCs from SCID patients, repairing the underlying genetic defect in these cells, and returning the repaired cells to the original host can replace the faulty immune system in SCID patients without the graft rejection or graft-versus-host disease that follows transplantation of cells from unrelated donors. Gaspar et al. do just that for two types of SCID: X-linked SCID (SCID-X1) and adenosine deaminase–deficient SCID (ADA-SCID). The authors repaired the underlying genetic defect in 10 of 10 patients with SCID-X1 and in 4 of 6 patients with ADA-SCID, resulting in the development of a functional polyclonal T cell repertoire that persisted for at least 9 years after therapy. The procedure produced minimal side effects and permitted all patients to attend typical schools. One patient in the SCID-X1 cohort developed a blood cancer, acute lymphoblastic leukemia (ALL), a complication observed in previous SCID-X1 gene therapy studies, but this patient is currently in remission. No cases of ALL developed in the ADA-SCID cohort. The promising results of these and similar studies, albeit with an increased risk of ALL in SCID-X1 patients, support the development of new safer and more efficient vectors for this and other kinds of gene therapy. Long-term follow-up of patient participants in early gene-therapy trials such as the ones described here is critical for scientists to decipher the parameters of success and failure for gene therapy in general—and for SCID-specific treatments to bubble over into the clinic. X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor γ chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)–matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34+ hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral immunity only partially recovered but was sufficient in some patients to allow for withdrawal of immunoglobulin replacement; however, three patients developed antibiotic-responsive acute pulmonary infection after discontinuation of antibiotic prophylaxis and/or immunoglobulin replacement. One patient developed acute T cell acute lymphoblastic leukemia because of up-regulated expression of the proto-oncogene LMO-2 from insertional mutagenesis, but maintained a polyclonal T cell repertoire through chemotherapy and entered remission. Therefore, gene therapy for SCID-X1 without myelosuppressive conditioning effectively restored T cell immunity and was associated with high survival rates for up to 9 years. Further studies using vectors designed to limit mutagenesis and strategies to enhance B cell reconstitution are warranted to define the role of this treatment modality alongside conventional HSCT for SCID-X1.