Alice F. Tarantal

Recombinant Adeno-Associated Virus Serotype 9 Leads to Preferential Cardiac Transduction In Vivo

Heart disease is often the end result of inherited genetic defects, which may potentially be treatable using a gene-transfer approach. Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a realistic method for the treatment of such disorders. Here, we demonstrate and compare the natural affinity of specific AAV serotype capsids for transduction of cardiac tissue. We compared the previously accepted optimal rAAV serotype for transduction of skeletal muscle, rAAV2/1, with rAAV2/8 and the newer rAAV2/9 vectors carrying the CMV-lacZ construct in their respective abilities to transcend vasculature and transduce myocardium following intravenous delivery of 1×1011 vector genomes in neonatal mice. We found that both rAAV2/8 and rAAV2/9 are able to transduce myocardium at ≈20- and 200-fold (respectively) higher levels than rAAV2/1. Biodistribution analysis revealed that rAAV2/9 and rAAV2/8 demonstrate similar behavior in extracardiac tissue. Vector genome quantification showed an increase in genome copy numbers in cardiac tissue for several weeks following administration, which corresponds to expression data. In addition, we intravenously administered 1×1011 vector genomes of rAAV2/9-CMV-lacZ into adult mice and achieved an expression biodistribution profile similar to that found following delivery to newborns. Although higher doses of virus will be necessary to approach those levels observed following neonatal injections, adult myocardium is also readily transduced by rAAV2/9. Finally, we have demonstrated physiological disease correction by AAV9 gene transfer in a mouse model of Pompe disease via ECG tracings and that intravenous delivery of the same vector preferentially transduces cardiac tissue in nonhuman primates.

Fetal, infant, adolescent and adult phenotypes of polycystic ovary syndrome in prenatally androgenized female rhesus monkeys.

Old World monkeys provide naturally occurring and experimentally induced phenotypes closely resembling the highly prevalent polycystic ovary syndrome (PCOS) in women. In particular, experimentally induced fetal androgen excess in female rhesus monkeys produces a comprehensive adult PCOS‐like phenotype that includes both reproductive and metabolic dysfunction found in PCOS women. Such a reliable experimental approach enables the use of the prenatally androgenized (PA) female rhesus monkey model to (1) examine fetal, infant and adolescent antecedents of adult pathophysiology, gaining valuable insight into early phenotypic expression of PCOS, and (2) to understand adult pathophysiology from a mechanistic perspective. Elevated circulating luteinizing hormone (LH) levels are the earliest indication of reproductive dysfunction in late gestation nonhuman primate fetuses and infants exposed to androgen excess during early (late first to second trimester) gestation. Such early gestation‐exposed PA infants also are hyperandrogenic, with both LH hypersecretion and hyperandrogenism persisting in early gestation‐exposed PA adults. Similarly, subtle metabolic abnormalities appearing in young nonhuman primate infants and adolescents precede the abdominal adiposity, hyperliplidemia and increased incidence of type 2 diabetes that characterize early gestation‐exposed PA adults. These new insights into the developmental origins of PCOS, and progression of the pathophysiology from infancy to adulthood, provide opportunities for clinical intervention to ameliorate the PCOS phenotype thus providing a preventive health‐care approach to PCOS‐related abnormalities. For example, PCOS‐like traits in PA monkeys, as in PCOS women, can improve with better insulin–glucose homeostasis, suggesting that lifestyle interventions preventing increased adiposity in adolescent daughters of PCOS mothers also may reduce their risk of acquiring many PCOS‐related metabolic abnormalities in adulthood. Am. J. Primatol. 71:776–784, 2009.

Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA

We developed a rapid and highly reproducible assay based on real-time PCR (TaqMan, Applied Biosystems, Foster City, CA) to quantitate simian immunodeficiency virus (SIV) RNA in plasma samples. This assay was compared with the current branched-chain DNA assay (Bayer, Emeryville, CA). Results obtained with the real-time TaqMan PCR assay were comparable to those obtained with the branched-chain DNA assay in overlapping ranges of sensitivities (r = 0.9429, p < 0.05). However, the real-time TaqMan PCR assay was capable of detecting as few as 50 copies of RNA/ml, whereas branched-chain DNA was only sensitive to 1,500 copies of RNA/ml. Therefore, several animals that tested negative by branched-chain DNA were positive by realtime TaqMan PCR. Two false positive tests were also recorded for the branched-chain DNA test. False negative and positive tests were confirmed by cell culture isolation and conventional nested RT-PCR. The SIV TaqMan assay detected a wide range of wild-type, cloned, and recombinant SIV strains with similar amplification efficiency, including SIVmac251, SIVmac239, SIVmac239 containing the 184V mutation in RT, SIV1A11, SIVmac239 delta3, SIVmac-M4, and chimeras (SHIVs) containing specific HIV-1 genes, such as reverse transcriptase (RT-SHIV) or Env (SHIV-E). In conclusion, the high sensitivity, increased specificity, wide dynamic range, simplicity, and reproducibility of the real-time SIV RNA quantitation allow the screening of large numbers of samples and make this method especially suitable for measuring both viral DNA and RNA levels during vaccine and therapy studies.

Ultrasound-induced lung hemorrhage in the monkey

Studies with the mouse have shown that lung hemorrhage can result from exposure to ultrasound at a peak pressure of approximately 1 MPa at 4 MHz (Mechanical Index [MI] approximately 0.5). In order to determine whether a comparable outcome could occur in a larger animal with characteristics similar to humans, studies were performed with monkeys using a clinical scanner under maximum output conditions (imaging + pulsed and color Doppler; derated pr of 3.7 MPa [4.5 MPa, measured in water], 4 MHz; MI approximately 1.8) (N = 57). Monkeys ranged in age from 1 day of life to 16 years with exposures limited to the right lung lobes (5 min cranial, 5 min caudal; N = 41 exposed, N = 12 sham-exposed controls, N = 4 colony controls). Results showed that animals ranging in age from 3 months to 5 years (mean age of 2.5 years) had a greater propensity for the occurrence of multiple well-demarcated circular hemorrhagic foci (0.1-1.0 cm), which were not observed in either control group. These lesions were characterized by marked congestion of alveolar capillaries with accumulation of red blood cells within the alveolar spaces, and were unassociated with major vessels or respiratory bronchioles. Further studies will be required in order to determine the relevance of these findings to the human, although it was concluded that ultrasound-induced lung hemorrhage in the monkey is of a significantly lesser degree when compared to the mouse.

Tissue Specificity of Decellularized Rhesus Monkey Kidney and Lung Scaffolds

Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.

Fetal and maternal outcome after administration of tenofovir to gravid rhesus monkeys (Macaca mulatta)

&NA; Tenofovir has been shown to cross the placenta in quantities sufficient to sustain reductions in viral load in simian immunodeficiency virus (SIV)‐infected fetal monkeys. With chronic exposure (30 mg/kg), however, significant bone‐related toxicity has been shown in ˜25% of infants studied. Further investigations were conducted to determine whether the bone‐related toxicity observed was initiated during fetal life. Gravid rhesus monkeys (n = 4) were administered tenofovir subcutaneously once daily from 20 to 150 days of gestation (30 mg/kg; term: 165 ± 10 days). Fetuses were monitored sonographically, and maternal and fetal blood and urine samples were collected to assess hematologic parameters, clinical chemistry, insulin‐like growth factor (IGF) levels, and bone biomarkers. Fetuses were delivered by hysterotomy near term for necropsy and evaluation of bone‐related mechanical properties. Results of these studies have shown 1) normal fetal development, although overall body weights and crown‐rump lengths were less than those for age‐matched controls (p ≤ .03); 2) a significant reduction in circulating IGF‐I (p < .001); 3) a small reduction in fetal bone porosity (p ≤ .03); and 4) transient alterations in maternal body weights and bone‐related biomarkers during the treatment period. The results of these studies suggest that chronic fetal exposure to tenofovir at the maternal dose of 30 mg/kg throughout gestation can alter select fetal parameters and transiently affect maternal bone biomarkers.

Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macaca mulatta)

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques offers opportunities to analyze mechanisms of CMV pathogenesis in a primate species. Four fetal rhesus monkeys were inoculated intraperitoneally with RhCMV early in the second trimester, and pregnancies were terminated by hysterotomy during the third trimester. Three fetuses had evidence of severe CMV disease, including intrauterine growth restriction, ventriculomegaly, microcephaly, lissencephaly, and extensive degenerative changes of the cerebral parenchyma. Histopathologic examination revealed polymicrogyria, gliosis, leptomeningitis, periventricular calcifications, and inclusion-bearing cells. These results demonstrate that the developing macaque brain is susceptible to infection with RhCMV early in the second trimester and that intrauterine infection results in neuropathologic outcomes similar to those observed in humans congenitally infected with CMV.

Renal Ontogeny in the Rhesus Monkey (Macaca mulatta) and Directed Differentiation of Human Embryonic Stem Cells Towards Kidney Precursors

The development of the metanephric kidney was studied immunohistochemically across gestation in monkeys to identify markers of cell specification, and to aid in developing experimental paradigms for renal precursor differentiation from human embryonic stem cells (hESC). PAX2, an important kidney developmental marker, was expressed at the tips of the ureteric bud, in the surrounding condensing mesenchyme, and in the renal vesicle. Vimentin, a mesenchymal and renal marker, was strongly expressed in the metanephric blastema then found to be limited to the glomerulus and interstitial cells of the medulla and cortex. A model of gene expression based on human and nonhuman primate renal ontogeny was developed and incorporated into studies of hESC differentiation. Spontaneous hESC differentiation revealed markers of metanephric mesenchyme (OSR1, PAX2, SIX2, WT1) that increased over time, followed by upregulation of kidney precursor markers (EYA1, LIM1, CD24). Directed hESC differentiation was also evaluated with the addition of retinoic acid, Activin-A, and BMP-4 or BMP-7, and using different culture substrate conditions. Of the culture substrates studied, gelatin most closely recapitulated the anticipated directed developmental pattern of renal gene expression. No differences were found when BMP-4 and BMP-7 were compared with baseline conditions. PAX2 and Vimentin immunoreactivity in differentiating hESC was also similar to the renal precursor patterns reported for human fetal kidneys and findings described in rhesus monkeys. The results of these studies are as follows: (1) provide additional data to support that rhesus monkey kidney development parallels that of humans, and (2) provide a useful model for hESC directed differentiation towards renal precursors.

Administration of 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) to gravid and infant rhesus macaques (Macaca mulatta): safety and efficacy studies.

9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) significantly inhibits viral reverse transcription and has been reported to sustain low virus load in SIV-infected rhesus monkeys. Based on these findings, studies were conducted to assess the safety, efficacy, and placental transfer of PMPA when administered once daily subcutaneously to gravid rhesus monkeys during the second and third trimesters and their offspring (30 mg/kg/day). Fetuses (SIV-infected, N = 6; noninfected, N = 6) were monitored sonographically, and maternal/fetal blood samples were collected at select time points for hematologic, clinical chemical, virologic, immunologic, and pharmacologic assessments. Newborns were delivered by cesarean section at term and nursery reared for postnatal studies. Infants were administered PMPA once daily beginning on day 2 of life until 9 months postnatal age. Results of these studies have shown significant placental transport of PMPA, with peak fetal levels at 1 to 3 hours post-maternal administration; a significant and sustained reduction in viral load in SIV-infected fetuses and infants; and marked improvements in outcome (e.g., survival, growth, health) in SIV-infected offspring. However, decreased infant body weights and alterations of select serum biochemical parameters (e.g., decreased phosphorus levels, elevated alkaline phosphatase) have been shown to occur in approximately 67% of PMPA-treated infants, with severe growth restriction and bone-related toxicity in approximately 25% of animals studied. These data suggest that although PMPA holds great promise for HIV-infected patients, there is the potential for bone-related toxicity at chronic, high dosages, particularly in infants.

Beta-Defensin 126 on the Surface of Macaque Sperm Mediates Attachment of Sperm to Oviductal Epithelia

Abstract Beta-defensin 126 (DEFB126) coats the entire surface of macaque sperm until sperm become capacitated, and the removal of DEFB126 from over the head of sperm is required for sperm-zona recognition. Viable sperm collected from cervix and the uterine lumen of mated female macaques had DEFB126 coating the entire surface, suggesting that DEFB126 is retained on sperm en route to the oviduct. DEFB126 plays a major role in attachment of sperm to oviductal epithelial cells (OECs). Following treatment to either remove or alter DEFB126, sperm were coincubated with explants of OECs, which were assessed for sperm binding following rinsing to remove superficially attached sperm. Sperm treated with either 1 mM caffeine + 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) (induces capacitation and complete release of DEFB126 from sperm), 2 mM caffeine (removes DEFB126 from over the head and midpiece but does not induce capacitation), anti-DEFB126 immunoglobulin, or neuraminidase (cleaves sialic acid from terminal positions on glycosylation sites of DEFB126) resulted in similar and significant levels of inhibition of sperm-OEC binding. Preincubation of OECs with soluble DEFB126 also resulted in significantly reduced sperm-OEC binding. Furthermore, reduced OEC binding capability of sperm lacking DEFB126 could be restored by addition of soluble DEFB126 to the sperm surface prior to incubation with OECs. Finally, purified DEFB126, infused into oviducts in situ, associated primarily with the apical membranes of secretory-type epithelial cells. In summary, treatments of macaque sperm that result in either removal, masking, or alteration of DEFB126 result in loss of sperm-OEC binding that is independent of changes in sperm motility. DEFB126 may be directly involved in the formation of a reservoir of sperm in the oviduct of macaques.