Single-dose MGTA-145/plerixafor leads to efficient mobilization and in vivo transduction of HSCs with thalassemia correction in mice

We have developed an in vivo hemopoietic stem cell (HSC) gene therapy approach without the need for myelosuppressive conditioning and autologous HSC transplantation. It involves HSC mobilization and IV injection of a helper-dependent adenovirus HDAd5/35++ vector system. The current mobilization regimen consists of granulocyte colony-stimulating factor (G-CSF) injections over a 4-day period, followed by the administration of plerixafor/AMD3100. We tested a simpler, 2-hour, G-CSF–free mobilization regimen using truncated GRO-β (MGTA-145; a CXCR2 agonist) and plerixafor in the context of in vivo HSC transduction in mice. The MGTA-145+plerixafor combination resulted in robust mobilization of HSCs. Importantly, compared with G-CSF+plerixafor, MGTA-145+plerixafor led to significantly less leukocytosis and no elevation of serum interleukin-6 levels and was thus likely to be less toxic. With both mobilization regimens, after in vivo selection with O6-benzylguanine (O6BG)/BCNU, stable GFP marking was achieved in >90% of peripheral blood mononuclear cells. Genome-wide analysis showed random, multiclonal vector integration. In vivo HSC transduction after mobilization with MGTA-145+plerixafor in a mouse model for thalassemia resulted in >95% human γ-globin+ erythrocytes at a level of 36% of mouse β-globin. Phenotypic analyses showed a complete correction of thalassemia. The γ-globin marking percentage and level were maintained in secondary recipients, further demonstrating that MGTA145+plerixafor mobilizes long-term repopulating HSCs. Our study indicates that brief exposure to MGTA-145+plerixafor may be advantageous as a mobilization regimen for in vivo HSC gene therapy applications across diseases, including thalassemia and sickle cell disease.

Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans

ABSTRACTIn prior studies of exocyst-mediated late secretion inCandida albicans, we have determined that Sec6 contributes to cell wall integrity, secretion, and filamentation. A conditional mutant lackingSEC6expression exhibits markedly reduced lateral hyphal branching. In addition, lack of the related t-SNAREs Sso2 and Sec9 also leads to defects in secretion and filamentation. To further understand the role of the exocyst in the fundamental processes of polarized secretion and filamentation inC. albicans, we studied the exocyst subunit Sec15. SinceSaccharomyces cerevisiae SEC15is essential for viability, we generated aC. albicansconditional mutant strain in whichSEC15was placed under the control of a tetracycline-regulated promoter. In the repressed state, cell death occurred after 5 h in the tetR-SEC15 strain. Prior to this time point, the tetR-SEC15 mutant was markedly defective in Sap and lipase secretion and demonstrated increased sensitivity to Zymolyase and chitinase. Notably, tetR-SEC15 mutant hyphae were characterized by a hyperbranching phenotype, in direct contrast to strain tetR-SEC6, which had minimal lateral branching. We further studied the localization of the Spitzenkörper, polarisomes, and exocysts in the tetR-SEC15 and tetR-SEC6 mutants during filamentation. Mlc1-GFP (marking the Spitzenkörper), Spa2-GFP (the polarisome), and Exo70-GFP (exocyst) localizations were normal in the tetR-SEC6 mutant, whereas these structures were mislocalized in the tetR-SEC15 mutant. Following alleviation of gene repression by removing doxycycline, first Spitzenkörper, then polarisome, and finally exocyst localizations were recovered sequentially. These results indicate that the exocyst subunits Sec15 and Sec6 have distinct roles in mediating polarized secretion and filamentation inC. albicans.

Evidence for Cellular Uptake and Subsequent Release of HIV-Derived Vector Particles after Ex Vivo Transduction Culture of Hematopoietic Cells.

Retroviral vectors based on Human Immunodeficiency Virus (HIV) and pseudotyped with vesicular stomatitis virus G (VSV-G) envelope glycoprotein can stably modify non-dividing hematopoietic stem cells. Ex vivo culture of vector particles and target cells is presumed to result in receptor mediated particle uptake by the cell and successful proviral integration, or degradation. However, recent evidence in dendritic cells suggests an alternate cellular fate for vector particles whereby they persist in exosomes and can be released to transduce secondary targets. We investigated the existence and consequences of such a pathway in other hematopoietic target cells after conventional ex vivo exposure. Murine bone marrow cells (5×10^5) were exposed to vector particles (2.5 ×10^6), washed twice, and placed alongside 293T fibroblasts (1×10^5). Direct, or transwell, co-culture resulted in GFP marking of 30% and 10% of secondary targets (ie. 293T cells), respectively. Transgene expression in 293T cells was stable over time in culture, abrogated by using integration-deficient particles, and reflected proviral integration, based on real-time PCR results. Cellular persistence of particles after primary exposure of murine whole bone marrow or SupT1 cells and secondary carryover were vector concentration dependent and pseudotype independent. Intriguingly, cell bound vector particles were selectively protected from serum inactivation, and the kinetics of secondary transduction suggested a prolonged particle half-life, when compared to particles cultured under cell-free conditions. Further, while direct protease exposure effectively eliminated vector infectivity, secondary transfer of particles from vector exposed, protease washed, cells was only partially inhibited, suggesting the uptake of particles in protease-inaccessible compartments of primary targets. When comparing vector exposures at 37 C versus 4 C (preventing particle uptake while permitting binding), again followed by protease treatment, secondary gene transfer was relatively greater at 37 C and increased with primary transduction duration, consistent with progressive intracellular particle retention. Indeed, deconvolution microscopy experiments to investigate the fate of tagged particles after brief vector exposure to target cells revealed intracellular persistence and perinuclear accumulation. To determine the relevance of these in vitro findings, we next performed studies in non-ablated murine recipients (n=30) that received ex vivo vector exposed, and washed, whole bone marrow or lineage depleted cells. To unambiguously demonstrate particle hand-off to recipient hematopoietic cells, we used CD45.1 donors and CD45.2 recipient animals. Results demonstrated long-term GFP marking by flow-cytometry (up to 9%) and by real-time PCR in recipient bone marrow and peripheral blood leukocytes. Additional immuno stains at sacrifice showed GFP-marked -CD45 negative- cells in liver and spleen tissue sections. Gain of replication competency in vector lots and in serum from recipient animals was excluded by p24 ELISA assay. Based on these results we propose an alternate fate for VSV-G lentivector particles, involving cellular retention after ex vivo exposure to primary hematopoietic target cells, subsequent release, and secondary transduction of susceptible target tissues. These findings may have implications for a range of applications using lentiviral vectors.

Multi-Copy Integration after HIV-Derived Lentivirus Vector Transduction of Murine Hematopoietic Stem Cells Does Not Promote Clonal Proliferation in Primary or Secondary Recipients.

Replication competent Moloney murine leukemia virus (MLV) retrovirus is used experimentally in murine models to identify potential oncogenes by repeat infection and resultant insertion of multiple proviral copies into the target cell genome. Mutagenicity in this system relies on proviral insertion in proximity of oncogenes, or their regulatory sequences leading to subsequent activation and clonal expansion. Replication incompetent MLV-derived vector particles are attractive vehicles for gene therapy strategies, and have been shown to retain these mutagenic properties after transduction of highly enriched murine and human hematopoietic progenitor and stem cells. Work by others suggests that this may in part occur in a vector particle dose and copy number dependent, transgene independent fashion (Modlich et al., Blood 2003 Suppl., abstract 699; Brugman et al., Molecular Therapy 2004 Suppl., abstract 1046). It is unclear if the same properties of mutagenic potential and copy number dependence apply to the insertion of HIV-derived lentivirus vectors. To address this question we have used VSV-G pseudotyped lentiviral vector (VSV-G/RRLsin-cPPThPGK-EGFPwpre) particles to transduce non-enriched mouse hematopoietic stem cells at escalating vector particle concentrations for subsequent repopulation of myeloablated murine recipients. Cells were transduced in culture with a single round of infection at a multiplicity of 1, 3, 10, or 30 in the presence of multiple cytokines and fibronectin fragment. Recipient animals readily reconstituted their hematopoietic system and demonstrated GFP marking in myeloid, B- and T- lymphoid cells. Peripheral blood counts, kinetics of GFP marking and flow-cytometric light scatter profiles showed no evidence of clonal proliferation. Primary recipients (n=23) were sacrificed between 5 and 7 months from transplantation to examine their marrow, peripheral blood and (in part) spleen for proviral marking by flow cytometry and real-time PCR. Results show a predicted increase in average proviral copy number ranging from 1.8 (range 1.2 – 2.8) after low MOI (1) infection to 17.5 (range 2.9 – 46.8) after high MOI (30) infection. Further, PCR amplification of genomic-proviral junction sites from progenitor colonies obtained at sacrifice of primary recipients showed no evidence of clonal restriction in the cohort of animals with an average of 7.9 proviral copies per GFP-marked cell (MOI 10 cohort). Secondary recipients (n=71) in all dose groups were followed for gene marking and, to date, have shown no signs of vector-driven clonal evolution. Our results suggest that HIV-derived lentivirus vector does not appear to have the same mutagenic properties at similar genomic copy numbers that others report for MLV-derived vectors in a comparable murine model system. This observation is consistent with distinct genomic insertion site preferences reported for HIV-derived vectors. We herein propose that, based on these and other advantageous features, lentivirus vectors are ideally suited for further pre-clinical and clinical exploration.

Direct Intramarrow Injection of CD34+ Cells May Improve Long-Term Engraftment in Nonhuman Primates.

Recent studies in the NOD/SCID model have shown improved engraftment of SCID-repopulating cells and higher levels of engraftment in the secondary transplantation when cells were administered by intramarrow (IM) versus intravenous (IV) injection suggesting that direct injection into the marrow cavity may be beneficial for stem cell engraftment in a clinical setting. To study whether IM injection was feasible and would result in improved engraftment in a clinically relevant large animal model, we compared IM vs IV injection in our competitive repopulation assay in baboons. Enriched CD34+ cells were split into 2 equal fractions and transduced with either a GFP- or YFP-expressing vector. Pretransplant transduction efficiencies and expansion of CD 34+ cells were similar in both fractions. One fraction was then infused into the marrow cavity of the right femur and the other fraction was given intravenously. Three baboons received gene-modified CD34+ enriched autologous bone marrow cells after myeloablative radiation. Peripheral blood granulocyte marking levels showed peaks at 2–3 weeks after transplantation and decreased thereafter. In all three monkeys, marking levels of IM injected cells (GFP) were lower than marking levels of IV injected cells (YFP) early after transplantation up to 7 weeks. However, in two of the three monkeys, GFP marking increased steadily after 2 months resulting in higher marking levels from IM injected cells. The trend sustained up to the last follow-up of nine months after transplantation, marking levels being 25.5% and 7.4% from IM and IV injected cells, respectively, in M00228. This pattern was recapitulated in the marking of bone marrow cells of the two animals. GFP (IM) and YFP (IV) marking levels of bone marrow cells from non-injected bone were 24.2% and 33.9%, respectively, at 1 month, 7.9% and 4.6% at 3 months, 19.1% and 12.6% at 6 months after transplantation in M00228. In addition, the GFP marking of the bone marrow cells from the injected bone was higher than that of the BM cells from non-injected bone while YFP marking level was similar. In conclusion, our data suggest that direct intramarrow injection of CD34+ cells may lead to improved engraftment of long-term repopulating cells. Clonal analysis is currently under way to determine the clonal pattern of the differentially marked repopulating cells.

Scaffold attachment region–containing retrovirus vectors improve long-term proviral expression after transplantation of GFP-modified CD34+ baboon repopulating cells

Sustained high-level proviral expression is important for clinical applications of gene therapy. Genetic elements including the β-interferon scaffold attachment region (SAR) have been shown to improve transgene expression in hematopoietic cells. We hypothesized that SAR elements might improve expression and allow the preselection of successfully transduced cells. Thus, we transplanted green fluorescent protein (GFP)–selected cells, half of which had been transduced with either SAR or non–SAR-containing retrovirus vectors, into 3 animals. All animals showed delayed engraftment compared with historic controls (28 vs 15.5 days). GFP marking was seen at levels up to 8% but declined over the first 6 weeks. Importantly, fluorescence intensity was 2- to 9-fold increased in progeny of SAR versus non–SAR vector–modified cells in all hematopoietic lineages for the duration of follow-up (6-12 months). In conclusion, the use of SAR-containing vectors improved transgene expression in hematopoietic repopulating cells, which may obviate the need for multicopy integration to achieve high-level expression and reduce the risk for insertional mutagenesis.