Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.

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.

Nonmyeloablative conditioning regimen to increase engraftment of gene-modified hematopoietic stem cells in young rhesus monkeys.

Immune responses to transgene products may lead to rejection of transduced cells, limiting successful gene therapy for genetic diseases. While moderate dosages of chemotherapeutic agents such as busulfan may increase hematopoietic stem cells (HSC) engraftment, they are not immune suppressive and do not abrogate immune responses to transgene products. Studies focused on nonmyeloablative conditioning with busulfan ± fludarabine in a clinically relevant monkey model to induce immune suppression to allow cells expressing a foreign transgene product to persist. Bone marrow CD34(+) HSC were transduced in two equal fractions using simian immunodeficiency virus (SIV)-based lentiviral vectors carrying a nonexpressed DNA sequence tag (NoN) and the green fluorescent protein (GFP) reporter gene. Post-transplant there was no evidence of elimination of cells containing the potentially immunogenic GFP gene; several recipients had stable persistence of cells, and no differences were detected with fludarabine, which was rapidly cleared. Antibodies and cellular immune responses to GFP developed in recipients with the highest levels of GFP-marked cells, although these cells were not eliminated. These studies establish a clinically relevant pediatric primate model to assess the effects of conditioning regimens on the engraftment of transduced HSC and the immune responses to cells expressing a foreign gene product.

Ultrafiltered recombinant AAV8 vector can be safely administered in vivo and efficiently transduces liver.

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 in vivo. 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.

Effects of busulfan dose escalation on engraftment of infant rhesus monkey hematopoietic stem cells after gene marking by a lentiviral vector.

OBJECTIVE: Non-myeloablative cytoreduction is used in clinical hematopoietic stem cell gene therapy trials to increase engraftment of gene-modified cells. We utilized an infant rhesus monkey model to identify an optimal dosage of busulfan that results in efficient long-term gene marking with minimal toxicities. METHODS: Bone marrow (BM) was harvested, followed by a single 2-hour intravenous infusion of busulfan at escalating dosages of 0 to 160 mg/m(2). CD34(+) cells were immunoselected from BM, transduced overnight with a simian immunodeficiency virus-based lentiviral vector carrying a non-expressed marker gene, and injected intravenously 48 hours post-busulfan administration. Pharmacokinetics were assessed, as well as adverse effects and peripheral blood and BM gene marking. RESULTS: Increasing dosages of busulfan resulted in increased area-under-the-curve (AUC) with some variability at each dosage level, suggesting interindividual variation in clearance. Blood chemistries were normal and no adverse effects were observed as a result of busulfan infusion. At 120 and 160 mg/m(2), transient neutropenia and thrombocytopenia were noted but not lymphopenia. Over the 6 months of study posttransplantation, a busulfan dosage-related increase in gene marking was observed ranging from undetectable (no busulfan) up to 0.1% gene-containing cells in animals achieving the highest busulfan AUC. This corresponds to a more than 100-fold increase in gene marking over the busulfan dosage range studied. CONCLUSIONS: These data indicate that increased gene marking of hematopoietic stem cells can be achieved by escalating busulfan dosages from 40 to 160 mg/m(2) without significant toxicity in infant nonhuman primates.

A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders.

Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes.

The spatiotemporal dynamics of triglyceride (TG) storage in unilocular adipocytes are not well understood. Here we applied ex vivo technology to study trafficking and metabolism of fluorescent fatty acids in adipose tissue explants. Live imaging revealed multiple cytoplasmic nodules surrounding the large central lipid droplet (cLD) of unilocular adipocytes. Each cytoplasmic nodule harbors a series of closely associated cellular organelles, including micro-lipid droplets (mLDs), mitochondria, and the endoplasmic reticulum. Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG. This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte. This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.

Potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28.

Adenovirus vaccine vectors derived from rare human serotypes have been shown to be less potent than serotype 5 (Ad5) at inducing immune responses to encoded antigens. To identify highly immunogenic adenovirus vectors, we assessed pro-inflammatory cytokine expression, binding to the CD46 receptor, and immunogenicity. Species D adenoviruses uniquely suppressed pro-inflammatory cytokines and induced high levels of type I interferon. Thus, it was unexpected that a vector derived from a representative serotype, Ad28, induced significantly higher transgene-specific T cell responses than an Ad35 vector. Prime-boost regimens with Ad28, Ad35, Ad14, or Ad5 significantly boosted T cell and antibody responses. The seroprevalence of Ad28 was confirmed to be <10% in the United States. Together, this shows that a rare human serotype-based vector can elicit strong immune responses, which was not predicted by in vitro results.

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.

Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus.

Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors. Both RRV and SFV glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that lentivirus vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping lentivirus vectors.