Adrian J. Thrasher

Effect of Gene Therapy on Visual Function in Leber's Congenital Amaurosis

Early-onset, severe retinal dystrophy caused by mutations in the gene encoding retinal pigment epithelium-specific 65-kD protein (RPE65) is associated with poor vision at birth and complete loss of vision in early adulthood. We administered to three young adult patients subretinal injections of recombinant adeno-associated virus vector 2/2 expressing RPE65 complementary DNA (cDNA) under the control of a human RPE65 promoter. There were no serious adverse events. There was no clinically significant change in visual acuity or in peripheral visual fields on Goldmann perimetry in any of the three patients. We detected no change in retinal responses on electroretinography. One patient had significant improvement in visual function on microperimetry and on dark-adapted perimetry. This patient also showed improvement in a subjective test of visual mobility. These findings provide support for further clinical studies of this experimental approach in other patients with mutant RPE65. (ClinicalTrials.gov number, NCT00643747 [ClinicalTrials.gov].).

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.

Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.

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.

WASP: a key immunological multitasker

The Wiskott–Aldrich syndrome protein (WASP) is an important regulator of the actin cytoskeleton that is required for many haematopoietic and immune cell functions, including effective migration, phagocytosis and immune synapse formation. Loss of WASP activity leads to Wiskott–Aldrich syndrome, an X-linked disease that is associated with defects in a broad range of cellular processes, resulting in complex immunodeficiency, autoimmunity and microthrombocytopenia. Intriguingly, gain of function mutations cause a separate disease that is mainly characterized by neutropenia. Here, we describe recent insights into the cellular mechanisms of these two related, but distinct, human diseases and discuss their wider implications for haematopoiesis, immune function and autoimmunity.

Clinical features and outcome of patients with IRAK-4 and MyD88 deficiency

Autosomal recessive interleukin-1 receptor-associated kinase (IRAK)-4 and myeloid differentiation factor (MyD)88 deficiencies impair Toll-like receptor (TLR)- and interleukin-1 receptor-mediated immunity. We documented the clinical features and outcome of 48 patients with IRAK-4 deficiency and 12 patients with MyD88 deficiency, from 37 kindreds in 15 countries. The clinical features of IRAK-4 and MyD88 deficiency were indistinguishable. There were no severe viral, parasitic, and fungal diseases, and the range of bacterial infections was narrow. Noninvasive bacterial infections occurred in 52 patients, with a high incidence of infections of the upper respiratory tract and the skin, mostly caused by Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The leading threat was invasive pneumococcal disease, documented in 41 patients (68%) and causing 72 documented invasive infections (52.2%). P. aeruginosa and Staph. aureus documented invasive infections also occurred (16.7% and 16%, respectively, in 13 and 13 patients, respectively). Systemic signs of inflammation were usually weak or delayed. The first invasive infection occurred before the age of 2 years in 53 (88.3%) and in the neonatal period in 19 (32.7%) patients. Multiple or recurrent invasive infections were observed in most survivors (n = 36/50, 72%). Clinical outcome was poor, with 24 deaths, in 10 cases during the first invasive episode and in 16 cases of invasive pneumococcal disease. However, no death and invasive infectious disease were reported in patients after the age of 8 years and 14 years, respectively. Antibiotic prophylaxis (n = 34), antipneumococcal vaccination (n = 31), and/or IgG infusion (n = 19), when instituted, had a beneficial impact on patients until the teenage years, with no seemingly detectable impact thereafter. IRAK-4 and MyD88 deficiencies predispose patients to recurrent life-threatening bacterial diseases, such as invasive pneumococcal disease in particular, in infancy and early childhood, with weak signs of inflammation. Patients and families should be informed of the risk of developing life-threatening infections; empiric antibacterial treatment and immediate medical consultation are strongly recommended in cases of suspected infection or moderate fever. Prophylactic measures in childhood are beneficial, until spontaneous improvement occurs in adolescence. Abbreviations: CRP = C-reactive protein, ELISA = enzyme-linked immunosorbent assay, IFN = interferon, IKBA = I&kgr;B&agr;, IL = interleukin, IL-1R = interleukin-1 receptor, InvBD = invasive bacterial disease, IRAK = interleukin-1 receptor-associated kinase, MyD = myeloid differentiation factor, NEMO = nuclear factor-kappaB essential modulator, NInvBD = noninvasive bacterial disease, TIR = Toll/IL-1R, TLR = Toll-like receptor, TNF = tumor necrosis factor.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.