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Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

Loss of monoubiquitination of histone H2B (H2Bub1) was found to be associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma. This study investigated the association and impact of the ubiquitin-specific peptidase 22 (USP22), an H2Bub1 deubiquitinase, on stem cell-like characteristics and cisplatin resistance in cancer-initiating cells (CIC) from primary lung adenocarcinoma. CICs were isolated, enriched, and characterized from patient-derived cancer tissues using both in vitro tumorsphere formation and in vivo xenograft assays. USP22 was determined to be predominantly expressed in CICs, a subpopulation of cells with high expression of the stem cell biomarkers, CD133 and CD44. The expression of USP22 in CICs is markedly reduced upon FBS/retinoic acid-induced differentiation. Moreover, knockdown of USP22 significantly suppressed tumorsphere formation and xenograft growth in NOD-SCID gamma (NSG) mice. Notably, USP22 and aldehyde dehydrogenase (ALDH) activity were elevated in tumorsphere cells that survived cisplatin treatment, whereas knockdown of USP22 significantly sensitizes tumorsphere cells to cisplatin. Interestingly, ALDH1A3, a predominant ALDH isozyme implicated in enhancing cisplatin resistance in lung adenocarcinoma, is significantly downregulated upon knockdown of USP22 in tumorsphere cells. Furthermore, knockdown of ALDH1A3 significantly sensitizes tumorsphere cells to cisplatin. Combined, these data demonstrate that USP22, predominantly expressed in CD133+ CICs, plays a critical role in tumorigenicity and cisplatin resistance in lung adenocarcinoma. Implications: Targeting USP22 represents a potential therapeutic approach to suppress CICs in lung adenocarcinoma partially through downregulation of ALDH1A3 expression. Mol Cancer Res; 16(7); 1161–71. ©2018 AACR.

Lentiviral vector (LVV)-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs) holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN) approximately 2-fold when added to mobilized peripheral blood (mPB) CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction, and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2) is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products. Keywords: lentiviral, HSPC, transduction

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with β-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs., Heffner et al. performed a small-molecule screen and identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Their data suggest that PGE2-mediated improvements in transduction efficiency may aid in clinical gene therapy applications using lentivirally modified HSPCs.

An important goal in gene therapy and stem cell transplant is to expand hematopoietic stem cells (HSCs) without causing hematopoietic abnormalities. This is particularly challenging in humans and other primates, where expansion protocols that work in mouse models are often less effective in primate HSCs. One approach is to test genes that have led to clonal expansions by vector insertion site effects in human gene therapy trials. In this study, we recreated a mutant HMGA2 cDNA that was associated with a benign clonal expansion in a patient treated on a β-thalassemia gene therapy trial. A lentiviral vector was generated that expressed a cDNA for human HMGA2 with a 3’ UTR deletion that eliminates all seven let-7 microRNA binding sites and thereby avoids the known repressive effects of let-7 miRNAs on HMGA2 expression. A gamma-retroviral MSCV promoter was used to express this mutant HMGA2 cDNA along with an Ires-GFP cassette. Bone marrow CD34+ cells were collected for Nemestrina Macaques and half the cells were transduced with the lentiviral vector containing the HMGA2-GFP construct while the other half was transduced with a mCherry control vector. The transduction efficiencies were approximately the same prior to transplant. At three months post transplantation, the marking in the peripheral blood mononuclear cells was 2.9% GFP+ and 1.1% mCherry+ for animal #16, and was 2.7% GFP+ and 3.2% mCherry+ for animal #27, suggesting relatively equivalent and low levels of transduction of repopulating cells. The HMGA2-GFP marking progressively increased over 21 and 26 months in the peripheral blood leukocytes to 39% for #16 and 41% for #27 while the mCherry marked cells have decreased (Fig 1AFig 1A). Equivalent levels of marking were seen in various mature peripheral blood lineages, including circulating erythyrocytes, suggesting that expansion had occurred in pluripotent HSCs. This HSC expansion was further demonstrated by a marking analysis in bone marrow cells that showed 44.6% and 75.2% GFP marking in the CD34+CD45RA- HSC compartment for animal #16 and #27 respectively, at the latest time point. Clonality analyses using vector integration sites (VIS) showed overall oligoclonal marking in both the GFP+ and mCherry+ cells in both animals (Fig 1BFig 1B). The top most frequent VISs were present in all PB lineages indicating that expansion had occurred in multiple HSC clones. The WBC counts, lineage distribution in the peripheral blood, the percentage of CD34+ cells in the bone marrow and all the mature lineages in the peripheral blood are all within the normal range, demonstrating lack of any detectable hematopoietic abnormality. Gene expression microarray analysis of RNA from sorted BM CD34+GFP+ and CD34+mCherry+ cells showed sharp upregulation of several genes in the HMGA2-expressing cells, particularly the IGF2BP gene, a known downstream target of HMGA2. In summary, our data show that long-term HSCs from non-human primates can be progressively expanded in vivo over several years by overexpressing HMGA2. This could be useful for gene therapy, particularly for expanding and obtaining high numbers of transduced erythrocytes and granulocytes for diseases in which a naturally occurring selection advantage is not present.View Large Image | Download PowerPoint Slide

Gene therapy for congenital hematopoietic disorders frequently relies on ex vivo lentiviral transduction of isolated CD34+ hematopoietic progenitor cells. Through a high-throughput small molecule screen, we identified PGE2 as a positive mediator of lentiviral transduction of hematopoietic stem and progenitor cells enriched from mobilized peripheral blood (PB CD34+ cells). CD34+ cells transduced with a VSVG-pseudotyped lentiviral vector in the presence of cytokines and 10 uM PGE2 yielded a vector copy number per cell (VCN) approximately 2-fold higher than CD34+ cells transduced in the absence of PGE2. This effect was seen consistently in 16 of 16 tested normal human donors in vitro, as well as primary CD34+ cells from both thalassemia and sickle cell disease patients. Importantly, PGE2 was observed to improve transduction of prospectively-isolated CD34+CD38- hematopoietic stem cells - a sub-population thought to be enriched for the long term repopulating stem cell. Transduction improvements were not associated with increased viral entry, but were associated with elevated expression of cAMP genes, supporting a post-entry mechanism of action that involves cAMP signaling downstream of prostaglandin receptors. Lastly, in a mouse xenotransplantation model of hematopoietic stem cell transplant, transduction of PB CD34+ cells in the presence of PGE2 improved VCN levels in engrafted human CD45+ cells 4-5 months post-transplant by ~2-fold without adversely affecting overall human cell engraftment. These data suggest that PGE2-mediated improvements in lentiviral transduction of human CD34+ cells could result in higher transduction efficiency and provide potential benefit in clinical gene therapy applications.

Lentiviral vector (LVV) mediated transduction of CD34+ hematopoietic stem and progenitor cells holds tremendous promise for the treatment of monogenic diseases of the blood. Critical to the success of this ex vivo gene therapy approach is the generation of a sufficient proportion of gene-modified cells. Here we investigated the potential of staurosporine, a protein kinase inhibitor, to enhance the transduction of LVVs in mobilized peripheral blood CD34+ cells both in vitro and in vivo. Staurosporine treatment has been previously demonstrated to increase HIV-1 integration in metaphase-arrested cells and shown to cause chromatin relaxation in metaphase cells (Manioui et al., Virology 2004). Additionally, in a separate study staurosporine treatment led to a 150% increase in transduction of CAp24+ CD4+ T cells (Permanyer et al., PLoS One 2013). Staurosporine increases vector copy number (VCN) approximately 2-fold when added to human mobilized peripheral blood (mPB) CD34+ enriched cells prior to transduction. The mechanism of this VCN improvement was investigated using the BLAM assay and a 1.5-fold improvement in viral entry was demonstrated. This effect was observed in at least six different mPB CD34+ cell lots that had various levels of transducibility. Interestingly, the effect of increased LVV transduction was most striking with low transducing cell lots and the effect diminished in higher transducing cell lots. Staurosporine treatment did not affect viability of cells post-transduction and there was no difference in in vitro colony formation in staurosporine-treated cells compared to vehicle-treated cells. NSG mice transplanted with cells transduced in the presence of 400 nM or 800 nM staurosporine demonstrated a statistically significant 3-fold and 4-fold increase in VCN in human CD45+ cells in bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Importantly, there was no significant difference in engraftment nor any observed lineage-bias between groups. Based on these observations, the use of staurosporine to enhance LVV transduction of human CD34+ cells is a promising method to improve the potential therapeutic benefit of gene therapy drug products.

Current manufacturing of clinical grade lentiviral vectors (LVVs) for gene therapy applications commonly relies on transient transfection of adherent 293T cells. Improvements in production efficiency and scalability would provide value in meeting the needs for increased amounts of vector required for clinical development. An inducible producer cell line grown in suspension culture represents a potentially more scalable manufacturing process for LVV production which eliminates the need for costly plasmid and transfection reagents. We have engineered a packaging cell line by introducing doxycycline-inducible Gag-Pol, Rev, and VSVG envelope genes into a suspension cell line. Packaging cell clones were isolated by single cell sorting and screened by qPCR for the presence of the delivered genes. Virus production was assessed by transient transfection of a lentiviral vector and doxycycline treatment. A titer of up to 5.0E6 transduction units per milliliter (TU/mL) was observed with packaging cell line stability demonstrated after greater than four months of continuous passage. Next, a self-inactivating lentiviral vector was excised from its plasmid backbone and ligated in vitro to a linear neomycin resistance cassette and used to transfect the lentiviral packaging cell line to generate a panel of producers. Following two weeks of G418 drug selection, adherent colonies were plucked and screened for viral production followed by single cell sorting to isolate individual producer clones. From five different plucked colonies with titers greater than 3.0E6 TU/mL, more than 100 clones were screened and 7 were identified that produced a harvest titer greater than 1.0E7 TU/mL. Four of these clones were successfully re-adapted to suspension and scaled up to 3L culture. LVV generated from individual producer clones was compared for the ability to transduce adult mobilized CD34+ hematopoietic stem cells (HSCs). Interestingly, the vector copy number (VCN) in CD34+ HSCs for the LVV derived from the producer cells varied between clones. Clones that produced the highest VCN have been chosen for further characterization in order to better understand the differences in HSC transduction. These lentiviral producer cell lines represent an important first step toward the creation of a manufacturing process that can better support clinical and commercial development of HSC lentiviral gene therapy.

Single-stranded DNA oligonucleotides (ODNs) can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, designed to direct a gene-editing event, into cells results in the activation of γH2AX, a well-recognized protein biomarker for double-stranded DNA breakage. Using a single copy, integrated mutant enhanced green fluorescent protein (eGFP) gene as our target, we now demonstrate that several types of ODNs, capable of directing gene editing, also activate the DNA damage response and the post-translational modification of proliferating cell nuclear antigen (PCNA), a signature modification of replication stress. We find that the gene editing reaction itself leads to transient DNA breakage, perhaps through replication fork collapse. Unmodified specific ODNs elicit a lesser degree of replication stress than their chemically modified counterparts, but are also less active in gene editing. Modified phosphothioate oligonucleotides (PTOs) are detrimental irrespective of the DNA sequence. Such collateral damage may prove problematic for proliferation of human cells genetically modified by gene editing.

Production of lentiviral vectors for clinical gene transfer trials is challenging due to the cost of acquiring GMP-grade plasmids and the labor intensive nature of making vectors via transient transfection. To facilitate clinical grade lentiviral vector production, we developed a packaging cell line, GPRG, which was used to make a fully stable producer, GPRG-EF1α-hgcOPT. This stable producer is presently supporting two clinical trials for the treatment of X-SCID. While initial results in one of these trials have been positive, the output and potency of this line mandates repeated production runs to supply enough vector for larger subjects, producing only enough for 2-3 cases per 6 day run. A second generation packaging cell line, GPRTG, has been developed and tested as a future platform for lentiviral gene transfer trials. This new cell line expresses codon-optimized HIV-1 gagpol, rev and tat. Expression of Rev, Tat and Vesicular Stomatitis Virus G proteins (VSV-G) are regulated by a Tet-Off/On system. We have improved the performance of the GPRTG cell line relative to GPRG by including the expression of HIV-1 Tat while decreasing the expression of Rev and by the independent re-insertion of the Rev, Tat and VSV-G packaging components. We generated a second fully stable producer, GPRTG-EF1α-hgcOPT, expressing the identical insulated SIN vector as in the first generation line. Both cell lines produce high titer vector preparations (>107 tu/ml) and are stable for at least 3 months of continuous passage. Although the vector titers are similar, GPRTG-EF1α-hgcOPT generated vector shows increased transduction efficiency of hCD34+ cells. PCR analysis of bulk CFU-GMs indicates a 2-fold increase in vector positive colonies and 3-5 fold increase in vector copy number with vector from GPRTG-EF1α-hgcOPT as compared to vector generated from GPRG-EF1α-hgcOPT when used at similar MOIs. We hypothesize that the basis for enhanced transduction efficiency of GPRTG derived vector is due to the expression of Tat and reduced expression of Rev each of which may affect vector transduction potency. In support of this hypothesis, our data show that transiently prepared vectors made with Rev only or in the presence of highly abundant Rev transduce hCD34+ cells less efficiently. We are currently employing the second generation cell line, GPRTG, to support future and on-going clinical protocols to treat other rare hematologic disorders including Wiskott-Aldrich Syndrome and Chronic Granulomatous Disease.