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A primary challenge in lentiviral gene therapy of β-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVβ-shα2, that allows coordinated expression of the therapeutic β A-T87Q -globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and β A-T87Q -globin gene expression identical to the parent vector. Compared with the first β A-T87Q -globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype., Competing Interests: Declaration of interests P.L. is a scientific founder and shareholder of bluebird bio, Inc. He is an inventor of awarded patents that cover the β(A-T87Q)-globin gene and the BB305 LentiGlobin vector., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the β AT87Q -globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure's suitability for human clinical applications while affording the additional safety of conditional suicide., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

A lentiviral vector encoding β-globin flanked by insulator elements has been used to treat β-thalassemia (β-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting β-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, β-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.

beta-Globin transgenes regulated by the locus control region (LCR) are dominantly silenced by linked bacterial reporter genes in transgenic mice. Enhanced green fluorescent protein (eGFP) from jellyfish is an alternative reporter used in retrovirus vectors to transfer LCRbeta-globin genes into bone marrow. We show here that the eGFP coding sequence silences LCRbeta-globin in transgenic mice, but the PGK promoter did not provoke such silencing. As eGFP contains 60 CpG dinucleotides, which are targets of DNA methylation, we synthesized a novel CpG-free variant called dmGFP. Its utility was demonstrated in MSCV retrovirus vectors transcriptionally controlled by the viral 5'LTR or internal PGK or EF1alpha promoter. Specific fluorescence was detected from eGFP, and at lower levels from dmGFP, in transduced mouse CFU-S and embryonic stem cells. While eGFP was rarely silenced in CFU-S, dmGFP was not silenced in these progenitors. Moreover, the dmGFP coding sequence did not silence LCRbeta-globin in transgenic mice, showing that the eGFP silencing mechanism acts primarily via CpG dinucleotides. However, LCRbeta-globin expression remained suboptimal, indicating that other silencing pathways recognize dmGFP in the absence of CpG dinucleotides. We conclude that dmGFP ameliorates silencing, but optimal LCRbeta-globin expression is obtained in the absence of nonmammalian reporters.

Clinical translation of gene-based therapies for arthritis could be accelerated by vectors capable of efficient intra-articular gene delivery and long-term transgene expression. Previously, we have shown that lentiviral vectors transduce rat synovium efficiently in vivo. Here, we evaluated the functional capacity of transgene expression provided by lentiviral-mediated gene delivery to the joint. To do this, we measured the ability of a lentiviral vector containing the cDNA for human interleukin-1 receptor antagonist (LV-hIL-1Ra) to suppress intra-articular responses to IL-1beta. Groups of rats were injected in one knee with 5 x 10(7) infectious units of LV-IL-1Ra. After 24 h, a range of doses of fibroblasts (3 x 10(3), 10(4), 3 x 10(4), or 10(5) cells) genetically modified to overexpress IL-1beta was injected into both knees. Intra-articular delivery of LV-hIL-1Ra strongly prevented swelling in all treated knees, even in those receiving the greatest dose of IL-1beta(+) cells. Cellular infiltration, cartilage erosion, and invasiveness of inflamed synovium were effectively prevented in LV-hIL-1Ra-treated knees and were significantly inhibited in contralateral joints. Beneficial effects were also observed systemically in the lentivirus-treated animals. Interestingly, intra-articular expression of the IL-1Ra transgene was found to increase in relation to the number of IL-1beta(+) cells injected. Further experiments using GFP suggest this is due to the proliferation of cells, stably modified by the integrative lentivirus, in response to inflammatory stimulation.

Retroviral transduction of hematopoietic stem cells (HSCs) offers an attractive strategy for treating malignancies that home to the marrow. This approach should therefore be of interest for evaluating the therapeutic activity of anti-angiogenic agents on hematopoietic malignancies whose growth has been associated with enhanced angiogenesis. A variety of studies have indicated endostatin to be a potent anti-angiogenic agent both in vitro and in vivo, and a human malignancy that might be sensitive to endostatin is human B-lineage acute lymphoblastic leukemia (B-ALL). The demonstrated ability of human B-ALL cells to engraft the marrow of immunodeficient mice suggested the potential of this system for testing an endostatin delivery strategy using co-transplanted non-obese diabetic-scid/scid (NOD/SCID) HSCs engineered to express endostatin. Here we show that, in spite of their mutant scid gene, NOD/SCID HSCs can be transduced with an endostatin-encoding retrovirus at efficiencies that result in a several-fold increase in endostatin serum levels in transplanted recipients. However, this did not alter the regrowth of co-transplanted human B-ALL blasts. These findings validate this gene transfer approach for investigating effects of novel therapeutics on primary human malignant cells that engraft NOD/SCID mice and question the utility of native endostatin for controlling human B-ALL in vivo.

Endostatin, a 20-kDa carboxy-terminal fragment of collagen XVIII, is the leading member of a class of physiologic inhibitors of angiogenesis with potent antitumor activity. Repeated subcutaneous administration of recombinant endostatin in mice led to permanent regression of established tumors to a microscopic dormant state and prompted the initiation of human clinical trials. However, a discrepancy remained unresolved: sustained tumor regression has only been observed with a non-soluble, precipitated form of recombinant endostatin produced in bacteria. To shed light on this question and establish a model of systemic anti-angiogenic gene therapy of cancer that may surmount obstacles in protein production and delivery, we transduced murine hematopoietic stem cells with a retrovirus encoding a secretable form of endostatin. Despite continuous, high-level secretion of endostatin in the vasculature of all transplanted mice, we detected neither inhibition of in vivo neoangiogenesis nor antitumor activity. Resolution of this paradox may come from human trials of endostatin now underway.

The delivery of anti-arthritic genes to the synovial lining of joints is being explored as a strategy for the treatment of rheumatoid arthritis. In this study, we have investigated the use of VSV-G pseudotyped, HIV-1-based lentiviral vectors for gene delivery to articular tissues. Recombinant lentivirus containing a beta-galactosidase/neomycin resistance fusion gene under control of the elongation factor (EF) 1alpha promoter efficiently transduced human and rat synoviocytes and chondrocytes in cell culture. When directly injected into the knees of rats, this vector transduced synovial lining cells, but not other articular tissues such as cartilage. We also constructed a lentiviral vector containing the human interleukin-1 receptor antagonist (IL1RA) cDNA and examined transgene expression in vitro and in vivo following injection into the knee joints of rats. In immunocompetent animals, intra-articular IL1RA expression was high and persisted, at a sharply declining rate, for approximately 20 days. In immunocompromised rats, however, lentivirus-mediated intra-articular expression of human IL1RA was found to persist for at least 6 weeks. Extra-articular expression of the transgene was minimal. These results indicate that lentiviral vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing long-term expression for gene-based treatments of arthritis.