Background In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. Methods We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. Results A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. Conclusions In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238 .).
Most patients with chronic lymphocytic leukemia (CLL) are elderly and/or have comorbidities that may make them ineligible for fludarabine-based treatment. For this population, chlorambucil monotherapy is an appropriate therapeutic option; however, response rates with chlorambucil are low, and more effective treatments are needed. This trial was designed to assess how the addition of rituximab to chlorambucil (R-chlorambucil) would affect safety and efficacy in patients with CLL.
Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium, CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.
Haemophilia B is an X-linked recessive bleeding disorder, arising from a deficiency of coagulation factor IX. It has been a target for gene therapy ever since the factor IX gene was cloned in 1982. Several distinct approaches have been evaluated in humans over the last 30 years, but none has resulted in tangible corrections of the bleeding phenotype in humans until recently. Our group has now shown that lasting clinical improvement of the bleeding phenotype in patients with haemophilia B is possible following a single systemic administration of a self-complementary adeno-associated virus vector to deliver an optimised factor IX expression cassette to the liver. Success in this trial raises hope for patients with severe haemophilia B as well as others with inherited monogenetic disorders of the liver where current treatment options are limited.
Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.
A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells.
Hemophilia B is a genetic disorder that is characterized by a deficiency of clotting factor IX (FIX) and excessive bleeding. Advanced understanding of the pathophysiology of the disease has led to the development of improved treatment strategies that aim to minimize the acute and long-term complications of the disease. Patients with hemophilia B are ideal candidates for gene therapy, mostly because a small increase in protein production can lead to significantly decreased bleeding diathesis. Although human clotting FIX was cloned and sequenced over 30 years ago, progress toward achieving real success in human clinical trials has been slow, with long-term, therapeutically relevant gene expression only achieved in one trial published in 2011. The history of this extensive research effort has revealed the importance of the interactions between gene therapy vectors and multiple arms of the host immune system at multiple stages of the transduction process. Different viral vector systems each have unique properties that influence their ability to deliver genes to different tissues, and the data generated in several clinical trials testing different vectors for hemophilia have guided our understanding toward development of optimal configurations for treating hemophilia B. The recent clinical success implementing a novel adeno-associated virus vector demonstrated sufficient FIX expression in patients to convert a severe hemophilia phenotype to mild, an achievement which has the potential to profoundly alter the impact of this disease on human society. Continued research should lead to vector designs that result in higher FIX activity at lower vector doses and with reduced host immune responses to the vector and the transgene product.
We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ? 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.
Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated virus has recently shown promise. This article will briefly outline a novel gene-transfer approach using self-complementary adeno-associated viral vectors using hemophilia B as a target disorder. This approach is currently being evaluated in the clinic. We will provide an overview of the development of self-complementary adeno-associated virus vectors as well as preclinical and clinical data with this vector system.
Intrauterine gene transfer (IUGT) offers ontological advantages including immune naiveté mediating tolerance to the vector and transgenic products, and effecting a cure before development of irreversible pathology. Despite proof-of-principle in rodent models, expression efficacy with a therapeutic transgene has yet to be demonstrated in a preclinical nonhuman primate (NHP) model. We aimed to determine the efficacy of human Factor IX (hFIX) expression after adeno-associated-viral (AAV)-mediated IUGT in NHP. We injected 1.0-1.95 × 10(13) vector genomes (vg)/kg of self-complementary (sc) AAV5 and 8 with a LP1-driven hFIX transgene intravenously in 0.9G late gestation NHP fetuses, leading to widespread transduction with liver tropism. Liver-specific hFIX expression was stably maintained between 8 and 112% of normal activity in injected offspring followed up for 2-22 months. AAV8 induced higher hFIX expression (P = 0.005) and milder immune response than AAV5. Random hepatocellular integration was found with no hotspots. Transplacental spread led to low-level maternal tissue transduction, without evidence of immunotoxicity or germline transduction in maternal oocytes. A single intravenous injection of scAAV-LP1-hFIXco to NHP fetuses in late-gestation produced sustained clinically-relevant levels of hFIX with liver-specific expression and a non-neutralizing immune response. These data are encouraging for conditions where gene transfer has the potential to avert perinatal death and long-term irreversible sequelae.
We previously showed that approximately 60% of B chronic lymphocytic leukaemia (B-CLL) cells express surface CD180, an orphan receptor of the Toll-like receptor family. Here we investigated the ability of anti-CD180 monoclonal antibody (mAb) to induce activation, cell cycling, survival and signalling in B-CLL cells and normal B cells. Upon addition of anti-CD180 mAb, alone or in combination with anti-CD40 mAb or recombinant IL-4 (rIL-4), expression of CD86, Ki-67, uptake of DiOC(6) , phosphorylation of signalling protein kinases and Ca(2+) flux were measured in B-CLL cells from untreated patients and normal B cells from age-matched volunteers. Normal B cells and approximately 50% of CD180(+) B-CLL clones responded to CD180 ligation by activation, cycling and increased survival comparable with, or superior to, those induced by anti-CD40 mAb or rIL-4 (Responder B-CLL). Non-responder CD180(+) B-CLL clones failed to respond to CD180 mAb and responded poorly to CD40 mAb and rIL-4. Anti-CD180 mAb induced phosphorylation of ZAP70/Syk, Erk, p38MAPK and Akt in normal B cells and Responder B-CLL cells. In contrast, Erk, p38MAPK and Akt were not phosphorylated in Non-responder B-CLL cells indicating a block in signalling and possible anergy. CD180 may provide powerful expansion and survival signals for Responder B-CLL cells and have an important prognostic value.
To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ?2?×?10(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ?20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24?hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.
Somatic in utero gene therapy aims to treat congenital diseases where pathology develops in perinatal life, thereby preventing permanent damage. The aim of this study was to determine whether delivery of self-complementary (sc) adeno-associated virus (AAV) vector in utero would provide therapeutic long-term transgene expression in a large animal model. We performed ultrasound-guided intraperitoneal injection of scAAV2/8-LP1-human Factor IX (hFIX)co (1 × 10(12) vector genomes/kg) in early (n = 4) or late (n = 2) gestation fetal sheep. The highest mean hFIX levels were detected 3 weeks after injection in late gestation (2,055 and 1,687.5 ng/ml, n = 2) and 3 days after injection in early gestation (435 ng/ml, n = 1). Plasma hFIX levels then dropped as fetal liver and lamb weights increased, although low levels were detected 6 months after late gestation injection (75 and 52.5 ng/ml, n = 2). The highest vector levels were detected in the fetal liver and other peritoneal organs; no vector was present in fetal gonads. hFIX mRNA was detectable only in hepatic tissues after early and late gestation injection. Liver function tests and bile acid levels were normal up to a year postnatal; there was no evidence of liver pathology. No functional antibodies to hFIX protein or AAV vector were detectable, although lambs mounted an antibody response after injection of hFIX protein and Freunds adjuvant. In conclusion, hFIX expression is detectable up to 6 months after delivery of scAAV vector to the fetal sheep using a clinically applicable method. This is the first study to show therapeutic long-term hFIX transgene expression after in utero gene transfer in a large animal model.
Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.
High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon (hIFN)-? delivered locally via gene transfer to effect vascular stabilization in an orthotopic model of glioblastoma xenograft resection.
Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.
Recent landmark studies show that it is now possible to convert somatic cells, such as skin fibroblasts and B lymphocytes, into pluripotent stem cells that closely resemble embryonic stem cells. These induced pluripotent stem (iPS) cells can be generated without using human embryos or oocytes, thus bypassing some of the ethical issues that have limited the use of human embryonic stems (hES) cells. Additionally, they can be derived from the patient to be treated, thereby overcoming problems of immunological rejection associated with the use of allogeneic hES cell derived progenitors. Whilst these patient-specific iPS cells have great clinical potential, their immediate utility is likely to be in drug screening and for understanding the disease process. This review discusses the promise of iPS cells as well as the challenges to their use in the clinic.
Ionizing radiation, an important component of glioma therapy, is critically dependent on tumor oxygenation. However, gliomas are notable for areas of necrosis and hypoxia, which foster radioresistance. We hypothesized that pharmacologic manipulation of the typically dysfunctional tumor vasculature would improve intratumoral oxygenation and, thus, the antiglioma efficacy of ionizing radiation.
Ionizing radiation is an important component of multimodal therapy for alveolar rhabdomyosarcoma (ARMS). We sought to evaluate the ability of IFN-beta to enhance the activity of ionizing radiation. Rh-30 and Rh-41 ARMS cells were treated with IFN-beta and ionizing radiation to assess synergistic effects in vitro and as orthotopic xenografts in CB17 severe combined immunodeficient mice. In addition to effects on tumor cell proliferation and xenograft growth, changes in the tumor microenvironment including interstitial fluid pressure, perfusion, oxygenation, and cellular histology were assessed. A nonlinear regression model and isobologram analysis indicated that IFN-beta and ionizing radiation affected antitumor synergy in vitro in the Rh-30 cell line; the activity was additive in the Rh-41 cell line. In vivo continuous delivery of IFN-beta affected normalization of the dysfunctional tumor vasculature of both Rh-30 and Rh-41 ARMS xenografts, decreasing tumor interstitial fluid pressure, increasing tumor perfusion (as assessed by contrast-enhanced ultrasonography), and increasing oxygenation. Tumors treated with both IFN-beta and radiation were smaller than control tumors and those treated with radiation or IFN-beta alone. Additionally, treatment with high-dose IFN-beta followed by radiation significantly reduced tumor size compared with radiation treatment followed by IFN-beta. The combination of IFN-beta and ionizing radiation showed synergy against ARMS by sensitizing tumor cells to the cytotoxic effects of ionizing radiation and by altering tumor vasculature, thereby improving oxygenation. Therefore, IFN-beta and ionizing radiation may be an effective combination for treatment of ARMS.
Resistance to angiogenesis inhibition can occur through the upregulation of alternative mediators of neovascularization. We used a combination of angiogenesis inhibitors with different mechanisms of action, interferon-beta (IFN-beta) and rapamycin, to target multiple angiogenic pathways to treat neuroblastoma xenografts.
We have previously shown an expansion of cytotoxic antigen-experienced CD4(+)T cells (CTLs) that express perforin (PF) in the peripheral blood of patients with B cell chronic lymphocytic leukaemia (B-CLL). Increased frequencies of CD4(+)CTLs have since been attributed to chronic viral infections, particularly, human cytomegalovirus (HCMV). The present study examined the involvement of CD4(+)CTLs in responses to HCMV in B-CLL, and characterized their differentiation. We studied 36 HCMV seropositive (SP) and seronegative B-CLL patients and 20 healthy age-matched individuals. The HCMV reactivity of CD4(+)PF(+) and CD4(+)PF(-) cells was determined by interferon-gamma expression, and expression of CD45RA and CCR7 was assessed by flow cytometry. Fluorescence in-situ hybridization was used to measure relative telomere lengths. CD4(+)PF(+)T cell expansion in B-CLL patients and controls was strongly associated with HCMV seropositivity. CD4(+)PF(+) compared to CD4(+)PF(-) cells from SP B-CLL patients elicited major histocompatibility complex (MHC) class II-restricted responses to HCMV. CD4(+)PF(+)T cells from patients and controls were enriched with highly differentiated T-effector/memory (CCR7(-)) and revertant (CCR7(-)CD45RA(+)) phenotype. CD4(+)PF(+)T cells from B-CLL patients had shorter telomeres than CD4(+)PF(-)T cells, indicating an extensive replicative history. We conclude that persistent exposure to HCMV antigens in SP B-CLL patients leads to an expansion of the circulating MHC class II-restricted CD4(+)PF(+)T cell population with effector/memory phenotype.
Hepatic gene transfer of atheroprotective human apoE by recombinant viral vectors can reverse hypercholesterolaemia and inhibit atherogenesis in apoE-deficient (apoE(-/-)) mice. Here, in preliminary studies we assess the effectiveness of a recently developed self-complementary adeno-associated virus (scAAV) serotype 8 vector, driven by a hepatocyte-specific promoter (LP1), for liver-directed gene delivery of human apoE3. Vector viability was validated by transducing cultured HepG2 cells and measuring secretion of apoE3 protein. Male and female apoE(-/-) mice, 6-month old and fed on normal chow, were intravenously injected with 1x10(11) vg (vector genomes) of scAAV2/8.LP1.apoE3; age-matched untreated mice served as controls. In male mice, plasma apoE3 levels were sufficiently high (up to 17 microg/ml) to normalize plasma total cholesterol and ameliorate their proatherogenic lipoprotein profile, by reducing VLDL/LDL and increasing HDL 5-fold. At termination (12 weeks) development of aortic atherosclerosis was significantly retarded by 58% (aortic lesion area 8.2+/-1.4% vs. 19.3+/-2.4% in control males; P<0.001). Qualitatively similar anti-atherogenic effects were noted when female mice were treated, but the benefits were less marked and aortic lesions, for example, were reduced by only 33% (15.7+/-3.7% vs. 23.6+/-6.9%). Although group numbers were small (n=4/5), this gender-specific difference reflected two to three times less apoE3 in plasma of female mice at weeks 3 and 6, implying that gene transfer to female liver using scAAV vectors may require additional optimization, despite their established superior potency to conventional single-stranded (ssAAV) vectors.
Dyskeratosis congenita (DC) is an inherited multi-system disorder characterised by muco-cutaneous abnormalities, bone marrow failure and a predisposition to malignancy. Bone marrow failure is the principal cause of mortality and is thought to be the result of premature cell death in the haematopoietic compartment because DC cells age prematurely and tend to have short telomeres. DC is genetically heterogeneous and patients have mutations in genes that encode components of the telomerase complex (DKC1, TERC, TERT, NOP10 and NHP2), and telomere shelterin complex (TINF2), both important in telomere maintenance. Here, we transduced primary T lymphocytes and B lymphocyte lines established from patients with TERC and DKC1 mutations with wild type TERC-bearing lentiviral vectors. We found that transduction with exogenous TERC alone was capable of increasing telomerase activity in mutant T lymphocytes and B lymphocyte lines and improved the survival and thus overall growth of B-lymphocyte lines over a prolonged period, regardless of their disease mutation. Telomeres in TERC-treated lines were longer than in the untreated cultures. This is the first study of its kind in DC lymphocytes and the first to demonstrate that transduction with TERC alone can improve cell survival and telomere length without the need for exogenous TERT.
Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are clinically heterogeneous and present as Leber Congenital Amaurosis, the severest form of early-onset retinal dystrophy and milder forms of retinal dystrophies such as juvenile retinitis pigmentosa and dominant cone-rod dystrophy. [Perrault, I., Rozet, J.M., Gerber, S., Ghazi, I., Leowski, C., Ducroq, D., Souied, E., Dufier, J.L., Munnich, A. and Kaplan, J. (1999) Leber congenital amaurosis. Mol. Genet. Metab., 68, 200-208.] Although not yet fully elucidated, AIPL1 is likely to function as a specialized chaperone for rod phosphodiesterase (PDE). We evaluate whether AAV-mediated gene replacement therapy is able to improve photoreceptor function and survival in retinal degeneration associated with AIPL1 defects. We used two mouse models of AIPL1 deficiency simulating three different rates of photoreceptor degeneration. The Aipl1 hypomorphic (h/h) mouse has reduced Aipl1 levels and a relatively slow degeneration. Under light acceleration, the rate of degeneration in the Aipl1 h/h mouse is increased by 2-3-fold. The Aipl1-/- mouse has no functional Aipl1 and has a very rapid retinal degeneration. To treat the different rates of degeneration, two pseudotypes of recombinant adeno-associated virus (AAV) exhibiting different transduction kinetics are used for gene transfer. We demonstrate restoration of cellular function and preservation of photoreceptor cells and retinal function in Aipl1 h/h mice following gene replacement therapy using an AAV2/2 vector and in the light accelerated Aipl1 h/h model and Aipl1-/- mice using an AAV2/8 vector. We have thus established the potential of gene replacement therapy in varying rates of degeneration that reflect the clinical spectrum of disease. This is the first gene replacement study to report long-term rescue of a photoreceptor-specific defect and to demonstrate effective rescue of a rapid photoreceptor degeneration.
Interferon-beta (IFN-beta) has been found to have anti-tumor properties against a variety of malignancies through different mechanisms. However, clinical trials involving systemic administration of IFN-beta have been hampered by secondary toxicity and the short half-life of IFN-beta in the circulation. In order to circumvent these limitations, we have developed an adeno-associated viral (AAV) vector gene-therapy approach to deliver IFN-beta to tumors. In this study, we tested the efficacy of AAV-mediated local delivery of IFN-beta for the treatment of retinoblastoma in preclinical models. Retinoblastoma is an ideal candidate for gene-therapy-based anti-cancer treatment because target cell transduction and, therefore, IFN-beta delivery can be contained within the ocular environment, thereby minimizing systemic toxicity. We report here that retinoblastoma cell lines exhibit pleiotropic responses to IFN-beta consistent with previous studies on a variety of tumor cell lines. Intravitreal injection of AAV-IFN-beta resulted in efficient retinal infection and sustained IFN-beta production in the eye with minimal systemic exposure. Vector spread outside of the eye was not detected. Using our orthotopic xenograft model of retinoblastoma, we found that intravitreal injection of AAV-IFN-beta had a potent anti-tumor effect in vivo. These data suggest that AAV-mediated delivery of IFN-beta may provide a complementary approach to systemic chemotherapy which is the standard of care for retinoblastoma around the world.
Single-gene disorders can cause perinatal mortality or severe permanent morbidity. Intrauterine gene therapy seeks to correct the genetic defect in the early stages of pathogenesis through delivery of a vector system expressing the therapeutic transgene to the fetus. Advantages of intrauterine gene therapy include prevention of irreversible organ damage, potentially inducing central tolerance and wider bio-distribution, including the brain after delivery of vector. Already, proof-of-cure has been demonstrated in knockout animal models for several diseases. Long-term outcomes pertaining to efficacy and durability of transgene expression and safety are under investigation in clinically relevant non-human primate models. Bystander effects in the mother from transplacental vector trafficking require further assessment. In this chapter, we discuss the candidate diseases amenable to intrauterine gene therapy, current state-of-the-art evidence, and potential clinical applications.
The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). The current method for eradicating inhibitors, termed immune tolerance induction (ITI), is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8) gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.
Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeutic gene transfer in extensive pre-clinical data and, recently, in clinical trials. Careful vector design and choice from a range of natural or synthetic pseudotypes allow targeted, efficient, and sustained expression of therapeutic genes. The efficiency of gene delivery can be further enhanced through the use of drug pre-treatment or co-infection with a suitable helper virus. This chapter describes current best practice for AAV production, including complete methods for: (1) efficient generation of vector without the use of helper viruses, simplifying the transition to GMP-grade production for clinical applications; (2) efficient and easily scalable purification of the virus by affinity chromatography, allowing rapid production of highly concentrated, high titre stocks; (3) reliable quantification and assaying of viral stocks, along with short- and long-term storage considerations.
Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat that connects the two monomer single-stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. In addition, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has low interassay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities.
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