Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the ß2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System.

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Elimination of the fibrinogen integrin αMß2-binding motif improves renal pathology in mice with sickle cell anemia.

Sickle cell anemia (SCA) is caused by a point mutation in the ß-globin gene that leads to devastating downstream consequences including chronic hemolytic anemia, episodic vascular occlusion, and cumulative organ damage resulting in death. SCA patients show coagulation activation and inflammation even in the absence of vascular occlusion. The coagulation factor fibrinogen is not only central to hemostasis but also plays important roles in pathologic inflammatory processes, in part by engaging neutrophils/macrophages through the αMß2 integrin receptor. To determine whether fibrin(ogen)-mediated inflammation is a driver of SCA-associated pathologies, hematopoietic stem cells from Berkeley sickle mice were transplanted into homozygous Fibγ390-396A mice that express normal levels of a mutant form of fibrin(ogen) that does not engage αMß2 Fibγ390-396A mice with SCA displayed an impressive reduction of reactive oxygen species (ROS) in white blood cells (WBCs), decreased circulating inflammatory cytokines/chemokines, and significantly improved SCA-associated glomerular pathology highlighted by reduced glomerulosclerosis, inflammatory cell infiltration, ischemic lesions, mesangial thickening, mesangial hypercellularity, and glomerular enlargement. In addition, Fibγ390-396A mice with SCA had improved glomerular protective responses and podocyte/mesangial transcriptional signatures that resulted in reduced albuminuria. Interestingly, the fibrinogen γ390-396A mutation had a negligible effect on cardiac, lung, and liver functions and pathologies in the context of SCA over a year-long observation period. Taken together, our data support that fibrinogen significantly contributes to WBC-driven inflammation and ROS production, which is a key driver of SCA-associated glomerulopathy, and may represent a novel therapeutic target against irreversible kidney damage in SCA.

High-titer foamy virus vector transduction and integration sites of human CD34(+) cell-derived SCID-repopulating cells.

Foamy virus (FV) vectors are promising tools for gene therapy, but low titer is a major challenge for large-scale clinical trials. Here, we increased FV vector titer 50-fold by constructing novel vector plasmids and using polyethylenimine-mediated transfection. FV and lentiviral (LV) vectors were used separately to transduce human CD34(+) cells at multiplicities of infection of 25, and those cells were transplanted into immunodeficient mice. FV vector transduction frequencies of repopulating human cells were 37.1 ± 1.9% in unstimulated cells and 36.9 ± 2.2% in prestimulated cells, and engraftment frequencies were 40.9 ± 4.9% in unstimulated cells and 47.1 ± 3.3% in prestimulated cells. Engraftment frequencies of FV vector-transduced cells were significantly higher than those of LV vector-transduced cells. Linear amplification-mediated PCR with Illumina paired-end runs showed that all human chromosomes contained FV provirus. FV had an integration preference near transcriptional start sites and CpG islands of RefSeq genes but not within genes. Repopulating lymphoid and myeloid cells contained common integration sites, suggesting that FV vector could transduce multilineage hematopoietic stem/progenitor populations. Our new FV vector backbone may be a suitable candidate for developing therapeutic FV vectors for use in clinical trials.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Hepatitis C virus stabilizes hypoxia-inducible factor 1alpha and stimulates the synthesis of vascular endothelial growth factor.

Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, which subsequently leads to hepatocellular carcinoma (HCC). The overexpression of the angiogenic factors has been demonstrated in HCC. In this study, we investigated the potential of HCV gene expression in inducing angiogenesis. Our results show that HCV infection leads to the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha). We further show that this stabilization was mediated via oxidative stress induced by HCV gene expression. The activation of NF-kappaB, STAT-3, PI3-K/AkT, and p42/44 mitogen-activated protein kinase was necessary for HIF-1alpha stabilization. HIF-1alpha induction in turn led to the stimulation of vascular endothelial growth factor. By using the chick chorioallantoic membrane assay, we show that HCV-infected cells released angiogenic cytokines, leading to neovascularization in vivo. These results indicate the potential of HCV gene expression in angiogenesis.

Eradication of Epstein-Barr virus episome and associated inhibition of infected tumor cell growth by adenovirus vector-mediated transduction of dominant-negative EBNA1.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1), a latent viral protein consistently expressed in infected proliferating cells, is essentially required in trans to maintain EBV episomes in cells. We constructed a mutant (mt) EBNA1 and examined whether it exerted dominant-negative effects on maintenance of the viral episome thereby leading to abrogation of EBV-infected tumor cell growth. Using lymphocyte and epithelial cell lines converted with neomycin-resistant recombinant EBV (rEBV) as models, adenovirus vector-mediated transduction of mtEBNA1, but not LacZ, brought about rapid and striking reductions in rEBV-derived wild-type EBNA1 levels and viral genomic loads in converted lines of three major viral latencies. This outcome was further validated at the single-cell level by cellular loss of G418 resistance and viral signals in situ. The mtEBNA1 transduction significantly impaired growth of naturally EBV-harboring Burkitt lymphoma cells in vitro and in vivo, largely in association with the eradication of viral episomes. Expression of mtEBNA1 per se caused no detectable cytotoxicity in EBV-uninfected cells. These results indicate that mtEBNA1 can act as a dominant-negative effector that efficiently impedes the EBV-dependent malignant phenotypes in cells regardless of viral latency or tissue origin. The mutant will afford an additional therapeutic strategy specifically targeting EBV-associated malignancies.