Lentiviral vectors pseudotyped with glycoproteins from Ross River and vesicular stomatitis viruses: variable transduction related to cell type and culture conditions

HIV-1-derived lentiviral vectors have been pseudotyped with various envelope glycoproteins to alter their host range. Previously, we found that envelope glycoproteins derived from the alphavirus Ross River virus (RRV) can pseudotype lentiviral vectors and mediate efficient transduction of a variety of epithelial and fibroblast-derived cell lines. In this study, we have investigated transduction of hematopoietic cells using RRV-pseudotyped vectors encoding the enhanced green fluorescent protein (EGFP). RRV-mediated transduction of human CD34+ cord blood cells and progenitors was very inefficient, even at multiplicities of infection of 100 (0.4% EGFP-positive progenitor colonies). Inefficient transduction was also observed in a variety of hematopoietic cell lines. However, two erythroleukemia-derived cell lines and monocytic cells that were driven to macrophage-like differentiation were moderately transduced. Transduction of hematopoietic cells with a control VSV-G-pseudotyped lentiviral vector was generally efficient, but unexpectedly decreased up to threefold upon stimulation of lymphocytic cell lines or primary murine bone marrow cells. Also, the tested hematopoietic cell lines were essentially nonpermissive for adeno-associated type 2 (AAV) vectors, and this was not affected by lineage, activity, or differentiation. Treatment of permissive 293 cells with proteases revealed that transduction with both the RRV- and the VSV-G-pseudotyped vectors in part depends on the presence of cell surface proteins. These results show a severely restricted ability of RRV glycoproteins to mediate transduction in hematopoietic cells that is likely due to specific receptor requirements that differ from those of VSV-G and AAV. Conversely, transduction with the VSV glycoprotein is affected by cellular activation more than widely believed. Our findings suggest that the envelope glycoproteins and culture conditions employed need to be carefully evaluated for each application. Furthermore, the uniquely restricted host range of RRV-pseudotyped vectors may aid in the design of novel cell-selective transduction strategies

Schematic comparison of the iodixanol purification (IP) and rapid purification (RP) methods.

<p>Depicted is a general overview of the steps required to produce, purify, and titer rAAV vectors using the RP and IP methods. F-T = Freeze-Thaw.</p

Ultrafiltered recombinant AAV8 vector can be safely administered <i>in vivo</i> and efficiently transduces liver

<div><p>Viral vectors are extensively purified for use in biomedical research, in order to separate biologically active virus particles and to eliminate production related impurities that are assumed to be detrimental to the host. For recombinant adeno-associated virus (rAAV) vectors this is typically accomplished using density gradient-based methods, which are tedious and require specialized ultracentrifugation equipment. In order to streamline the preparation of rAAV vectors for pilot and small animal studies, we recently devised a simple ultrafiltration approach that permits rapid virus concentration and partial removal of production-related impurities. Here we show that systemic administration of such rapidly prepared (RP) rAAV8 vectors in mice is safe and efficiently transduces the liver. Across a range of doses, delivery of RP rAAV8-CMV-eGFP vector induced enhanced green fluorescent protein (eGFP) expression in liver that was comparable to that obtained from a conventional iodixanol gradient-purified (IP) vector. Surprisingly, no liver inflammation or systemic cytokine induction was detected in RP rAAV injected animals, revealing that residual impurities in the viral vector preparation are not deleterious to the host. Together, these data demonstrate that partially purified rAAV vector can be safely and effectively administered <i>in vivo</i>. The speed and versatility of the RP method and lack of need for cumbersome density gradients or expensive ultracentrifuge equipment will enable more widespread use of RP prepared rAAV vectors, such as for pilot liver gene transfer studies.</p></div

eGFP transgene expression is comparable between IP and RP preparation methods.

<p>Liver tissue was obtained from 129/S mice injected intraperitoneally with PBS, IP-rAAV8-CMV-eGFP, or RP-rAAV8-CMV-eGFP. Indicated doses for rAAV8 vectors are 0.75 x 10¹¹ vg/mouse (Low), 2.25 x 10¹¹ vg/mouse (Mid), and 7.50 x 10¹¹ vg/mouse (High). <b>(A)</b> Liver tissue lysate was analyzed by anti-GFP immunoblotting. Anti-<i>β</i> actin immunoblotting is shown as a loading control. <b>(B)</b> Isolated liver RNA was analyzed by quantitative RT-PCR for eGFP transcript level. Expression is represented as relative to the 0.75 x 10¹¹ vg RP-rAAV8-CMV-eGFP data set (<i>β</i> actin-normalized). Data are represented as scatter plot with mean ± S.E.M. bars (n = 3 to 6 mice). Unpaired t-tests with Welch’s correction were performed to determine p-values.</p

Injection with RP-rAAV8-CMV-eGFP does not induce expression of inflammatory markers in mouse liver or systemic cytokines.

<p>129/S mice were injected intraperitoneally with PBS, IP-rAAV8-CMV-eGFP, or RP-rAAV8-CMV-eGFP. <b>(A)</b> Isolated liver RNA was used for quantitative RT-PCR using primers for TNFα, IL-6, Activin B, or Hepcidin. Expression is represented as relative to the PBS data set (<i>β</i> actin-normalized). Data are represented as scatter plot with mean ± S.E.M. bars (n = 3 to 6 mice). One-way ANOVA with Dunnett’s correction did not reveal any significant differences in the means of treatment groups compared to the PBS control group. Indicated doses for rAAV8 vectors are 0.75 x 10¹¹ vg/mouse (Low), 2.25 x 10¹¹ vg/mouse (Mid), or 7.50 x 10¹¹ vg/mouse (High). <b>(B)</b> Serum cytokine concentrations before and after rAAV injection with 7.50 x 10¹¹ vg/mouse were measured by immunoassay. Concentrations units are represented as mean ± S.E.M. (n = 4 mice). Multiple comparison of means using two-way ANOVA with Bonferroni correction did not detect any statistically significant differences between PBS and the treatment groups at each time point. No detectable expression was observed for the cytokines GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IP-10, KC, MCP-1, MIG, MIP-1α, and TNF-α.</p

Injection of IP and RP rAAV8-CMV-eGFP vectors induce dose-dependent neutralizing antibody responses.

<p>ID₅₀ neutralizing antibody assays were carried out on sera of mice that were injected with a 0.75 x 10¹¹ vg/mouse (Low), 2.25 x 10¹¹ vg/mouse (Mid), or 7.50 x 10¹¹ vg/mouse (High) dose of IP or RP rAAV8-CMV-eGFP. Data are represented as scatter plot of neutralizing anti-AAV8 response titers (ID₅₀) with mean ± S.E.M. bars (n = 3 to 6 mice). Unpaired t-tests with Welch’s correction were performed to determine p-values. Pre-injection, all mice in the rAAV treatment groups had a negligible anti-AAV8 response (titer <1:50).</p

Prophylaxis of invasive fungal infections in patients with hematological malignancies and solid tumors - Guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO)

Morbidity and mortality in patients with malignancies, especially leukemia and lymphoma, are increased by invasive fungal infections. Since diagnosis of invasive fungal infection is often delayed, antifungal prophylaxis is an attractive approach for patients expecting prolonged neutropenia. Antifungal prophylaxis has obviously attracted much interest resulting in dozens of clinical trials since the late 1970s. The non-absorbable polyenes are probably ineffective in preventing invasive fungal infections, but may reduce superficial mycoses. Intravenous amphotericin B and the newer azoles were used in clinical trials, but their role in antifungal prophylaxis is still not well defined. Allogeneic stem cell transplant recipients are at particularly high risk for invasive fungal infections. Other well described risk factors are neutropenia >10 days, corticosteroid therapy, sustained immunosuppression, graft versus host disease, and concomitant viral infections. The enormous study efforts are contrasted by a scarcity of risk stratified evidence based recommendations for clinical decision making. The objective of this review accumulating information on about 10.000 patients is to assess evidence based criteria primarily regarding the efficacy of antifungal prophylaxis in neutropenic cancer patients

Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options