AAV8 gene therapy reverses cardiac pathology and prevents early mortality in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of the cardiac phenotype of FRDA was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxnflox/null::MCK-Cre conditional knockout mouse (FXN-MCK) has an FXN gene ablation that prevents FXN expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss, and morbidity. FXN-MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN genes under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN-MCK control mice, AAV-treated FXN-MCK mice displayed increases in body weight, reversal of cardiac deficits, and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks postdose. FXN protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy.

High-throughput transcriptome analyses from ASPIRO, a phase 1/2/3 study of gene replacement therapy for X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level.

Development of Hematopoietic Stem Cell-Engineered Invariant Natural Killer T Cell Therapy for Cancer.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.

Pre-clinical Development of a Lentiviral Vector Expressing the Anti-sickling ßAS3 Globin for Gene Therapy for Sickle Cell Disease.

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) ß-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a ß-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the ß-globin promoter and a reduced version of the ß-globin locus-control region. The vector (GLOBE-AS3) transduced 60%-80% of mobilized CD34+ hematopoietic stem-progenitor cells (HSPCs) and drove ßAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12-14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.

Gene Therapy for Sickle Cell Disease: A Lentiviral Vector Comparison Study.

Sickle cell disease (SCD) is an inherited blood disorder caused by a single amino acid substitution in the ß-globin chain of hemoglobin. Gene therapy is a promising therapeutic alternative, particularly in patients lacking an allogeneic bone marrow (BM) donor. One of the major challenges for an effective gene therapy approach is the design of an efficient vector that combines high-level and long-term ß-globin expression with high infectivity in primary CD34+ cells. Two lentiviral vectors carrying an anti-sickling ß-globin transgene (AS3) were directly compared: the Lenti/ßAS3-FB, and Globe-AS3 with and without the FB insulator. The comparison was performed initially in human BM CD34+ cells derived from SCD patients in an in vitro model of erythroid differentiation. Additionally, the comparison was carried out in two in vivo models: First, an NOD SCID gamma mouse model was used to compare transduction efficiency and ß-globin expression in human BM CD34+ cells after transplant. Second, a sickle mouse model was used to analyze ß-globin expression produced from the vectors tested, as well as hematologic correction of the sickle phenotype. While minor differences were found in the vectors in the in vitro study (2.4-fold higher vector copy number in CD34+ cells when using Globe-AS3), no differences were noted in the overall correction of the SCD phenotype in the in vivo mouse model. This study provides a comprehensive in vitro and in vivo analysis of two globin lentiviral vectors, which is useful for determining the optimal candidate for SCD gene therapy.

Preclinical studies for a phase 1 clinical trial of autologous hematopoietic stem cell gene therapy for sickle cell disease.

BACKGROUND AIMS: Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials. METHODS: Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/ßAS3-FB vector and cryopreserving CD34+ cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/ßAS3-FB LV in the context of a murine BM transplant. RESULTS: Primary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/ßAS3-FB vector in cells from primary and secondary transplanted mice. CONCLUSIONS: We present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD.

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells.

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 108 to 1 × 109 CD34+ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34+ cells uses LV inefficiently, because the majority of CD34+ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34+CD38- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34+CD38- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34+ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34+CD38- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38+ cells. Importantly, LV modification and transplantation of IB-purified CD34+CD38- cells/non-modified CD38+ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34+ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the ß-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

Targeted genome editing technology can correct the sickle cell disease mutation of the ß-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the ß-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

The human ankyrin 1 promoter insulator sustains gene expression in a ß-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the ß-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human ß-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term ß-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the ß-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the ß-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.

Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells.

Sickle cell disease (SCD) can be cured by allogeneic hematopoietic stem cell transplant. However, this is only possible when a matched donor is available, making the development of gene therapy using autologous hematopoietic stem cells a highly desirable alternative. We used a culture model of human erythropoiesis to directly compare two insulated, self-inactivating, and erythroid-specific lentiviral vectors, encoding for γ-globin (V5m3-400) or a modified ß-globin (ßAS3-FB) for production of antisickling hemoglobin (Hb) and correction of red cell deformability after deoxygenation. Bone marrow CD34+ cells from three SCD patients were transduced using V5m3-400 or ßAS3-FB and compared with mock-transduced SCD or healthy donor CD34+ cells. Lentiviral transduction did not impair cell growth or differentiation, as gauged by proliferation and acquisition of erythroid markers. Vector copy number averaged approximately one copy per cell, and corrective globin mRNA levels were increased more than sevenfold over mock-transduced controls. Erythroblasts derived from healthy donor and mock-transduced SCD cells produced a low level of fetal Hb that was increased to 23.6 ± 4.1% per vector copy for cells transduced with V5m3-400. Equivalent levels of modified normal adult Hb of 17.6 ± 3.8% per vector copy were detected for SCD cells transduced with ßAS3-FB. These levels of antisickling Hb production were sufficient to reduce sickling of terminal-stage red blood cells upon deoxygenation. We concluded that the achieved levels of fetal Hb and modified normal adult Hb would likely prove therapeutic to SCD patients who lack matched donors.

Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by ß-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-ß(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy.

ß-globin gene transfer to human bone marrow for sickle cell disease.

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-ßAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34+ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-ßAS3-FB LV transduced BM CD34+ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total ß-globin-like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34+ cells were transplanted into immunodeficient mice, and the human cells recovered after 2-3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34+ cells that were directly differentiated in vitro. These results demonstrate that the CCL-ßAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling ß-globin gene in human SCD BM CD34+ progenitor cells, improving physiologic parameters of the resulting red blood cells.

The 3' region of the chicken hypersensitive site-4 insulator has properties similar to its core and is required for full insulator activity.

Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the "core", which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 5' core with a novel 3' 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.

Genotoxic potential of lineage-specific lentivirus vectors carrying the beta-globin locus control region.

Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction.

We show that lentiviral delivery of human gamma-globin gene under beta-globin regulatory control elements in hematopoietic stem cells (HSCs) results in sufficient postnatal fetal hemoglobin (HbF) expression to correct sickle cell anemia (SCA) in the Berkeley "humanized" sickle mouse. Upon de-escalating the amount of transduced HSCs in transplant recipients, using reduced-intensity conditioning and varying gene transfer efficiency and vector copy number, we assessed critical parameters needed for correction. A systematic quantification of functional and hematologic red blood cell (RBC) indices, organ pathology, and life span was used to determine the minimal amount of HbF, F cells, HbF/F-cell, and gene-modified HSCs required for correcting the sickle phenotype. We show that long-term amelioration of disease occurred (1) when HbF exceeded 10%, F cells constituted two-thirds of the circulating RBCs, and HbF/F cell was one-third of the total hemoglobin in sickle RBCs; and (2) when approximately 20% gene-modified HSCs repopulated the marrow. Moreover, we show a novel model using reduced-intensity conditioning to determine genetically corrected HSC threshold that corrects a hematopoietic disease. These studies provide a strong preclinical model for what it would take to genetically correct SCA and are a foundation for the use of this vector in a human clinical trial.

Mechanism of reduction in titers from lentivirus vectors carrying large inserts in the 3'LTR.

Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.

Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy.

Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.