Claire A. Schreiber

An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

B-Type Natriuretic Peptide Deletion Leads to Progressive Hypertension, Associated Organ Damage, and Reduced Survival: Novel Model for Human Hypertension

Altered myocardial structure and function, secondary to chronically elevated blood pressure, are leading causes of heart failure and death. B-type natriuretic peptide (BNP), a guanylyl cyclase A agonist, is a cardiac hormone integral to cardiovascular regulation. Studies have demonstrated a causal relationship between reduced production or impaired BNP release and the development of human hypertension. However, the consequences of BNP insufficiency on blood pressure and hypertension-associated complications remain poorly understood. Therefore, the goal of this study was to create and characterize a novel model of BNP deficiency to investigate the effects of BNP absence on cardiac and renal structure, function, and survival. Genetic BNP deletion was generated in Dahl salt-sensitive rats. Compared with age-matched controls, BNP knockout rats demonstrated adult-onset hypertension. Increased left ventricular mass with hypertrophy and substantially augmented hypertrophy signaling pathway genes, developed in young adult knockout rats, which preceded hypertension. Prolonged hypertension led to increased cardiac stiffness, cardiac fibrosis, and thrombi formation. Significant elongation of the QT interval was detected at 9 months in knockout rats. Progressive nephropathy was also noted with proteinuria, fibrosis, and glomerular alterations in BNP knockout rats. End-organ damage contributed to a significant decline in overall survival. Systemic BNP overexpression reversed the phenotype of genetic BNP deletion. Our results demonstrate the critical role of BNP defect in the development of systemic hypertension and associated end-organ damage in adulthood.

B-Type Natriuretic Peptide Deletion Leads to Progressive Hypertension, Associated Organ Damage, and Reduced SurvivalNovelty and Significance: Novel Model for Human Hypertension

Altered myocardial structure and function, secondary to chronically elevated blood pressure, are leading causes of heart failure and death. B-type natriuretic peptide (BNP), a guanylyl cyclase A agonist, is a cardiac hormone integral to cardiovascular regulation. Studies have demonstrated a causal relationship between reduced production or impaired BNP release and the development of human hypertension. However, the consequences of BNP insufficiency on blood pressure and hypertension-associated complications remain poorly understood. Therefore, the goal of this study was to create and characterize a novel model of BNP deficiency to investigate the effects of BNP absence on cardiac and renal structure, function, and survival. Genetic BNP deletion was generated in Dahl salt-sensitive rats. Compared with age-matched controls, BNP knockout rats demonstrated adult-onset hypertension. Increased left ventricular mass with hypertrophy and substantially augmented hypertrophy signaling pathway genes, developed in young adult knockout rats, which preceded hypertension. Prolonged hypertension led to increased cardiac stiffness, cardiac fibrosis, and thrombi formation. Significant elongation of the QT interval was detected at 9 months in knockout rats. Progressive nephropathy was also noted with proteinuria, fibrosis, and glomerular alterations in BNP knockout rats. End-organ damage contributed to a significant decline in overall survival. Systemic BNP overexpression reversed the phenotype of genetic BNP deletion. Our results demonstrate the critical role of BNP defect in the development of systemic hypertension and associated end-organ damage in adulthood.

Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration

Human induced pluripotent stem cells (iPSCs) and derived progeny provide invaluable regenerative platforms, yet their clinical translation has been compromised by their biosafety concern. Here, we assessed the safety of transplanting patient‐derived iPSC‐generated pancreatic endoderm/progenitor cells. Transplantation of progenitors from iPSCs reprogrammed by lentiviral vectors (LV‐iPSCs) led to the formation of invasive teratocarcinoma‐like tumors in more than 90% of immunodeficient mice. Moreover, removal of primary tumors from LV‐iPSC progeny‐transplanted hosts generated secondary and metastatic tumors. Combined transgene‐free (TGF) reprogramming and elimination of residual pluripotent cells by enzymatic dissociation ensured tumor‐free transplantation, ultimately enabling regeneration of type 1 diabetes‐specific human islet structures in vivo. The incidence of tumor formation in TGF‐iPSCs was titratable, depending on the oncogenic load, with reintegration of the cMYC expressing vector abolishing tumor‐free transplantation. Thus, transgene‐free cMYC‐independent reprogramming and elimination of residual pluripotent cells are mandatory steps in achieving transplantation of iPSC progeny for customized and safe islet regeneration in vivo.

Prognostic value of discs large homolog 7 transcript levels in prostate cancer.

Hypoxia has been associated with malignant progression, metastasis and resistance to therapy. Hence, we studied expression of hypoxia–regulated genes in 100 prostate cancer (CaP) bulk tissues and 71 adjacent benign tissues. We found 24 transcripts significantly overexpressed (p≤0.02). Importantly, higher transcript levels of disc large (drosophila) homolog-associated protein 5 (DLGAP5)/discs large homolog 7 (DLG7)/hepatoma up-regulated protein (HURP), hyaluronan-mediated motility receptor (HMMR) and cyclin B1 (CCNB1) were associated with higher Gleason score and more advanced systemic progression. Since the products of HMMR and CCNB1 have been identified recently as molecular markers of CaP progression, we postulated that DLG7 has prognostic value too. To test this hypothesis, we measured transcript levels for DLG7 in a 150-pair case-control cohort. The cases (progression to systemic disease within six years of surgery) and controls (no progression within eight years) were matched for clinical and pathologic prognostic variables, including grade, stage, and preoperative serum levels of PSA. The overall prognostic ability of DLG7, as tested in receiver operating characteristic analysis was of 0.74 (95% CI, 0.68 to 0.8). Overall, our data indicate that expression of DLG7, a hypoxia-controlled gene, holds prognostic potential in high-risk CaP; this also demonstrates that variation of oxygen tension may constitute a tool for identification of novel biomarkers for CaP.

Sustained ELABELA Gene Therapy in High-salt Diet-induced Hypertensive Rats

BACKGROUND Elabela (ELA) is a recently identified apelin receptor agonist essential for cardiac development, but its biology and therapeutic potential are unclear. In humans, ELA transcripts are detected in embryonic stem cells, induced pluripotent stem cells, kidney, heart and blood vessels. ELA through the apelin (APJ) receptor promotes angiogenesis in vitro, relaxes murine aortic blood vessels and attenuates high blood pressure in vivo. The APJ receptor when bound to its original ligand, apelin, exerts peripheral vasodilatory and positive inotropic effects, conferring cardioprotection in vivo. METHODS This study initially assessed endogenous ELA expression in normal and diseased rats and then characterized the effects of long-term ELA gene delivery by adeno-associated virus serotype 9 (AAV9) vectors on cardiorenal function in Dahl salt-sensitive rats (DS) on a high-salt diet over 3 months. RESULTS Endogenous ELA was predominantly expressed in the kidneys, especially in the renal collecting duct cells and was not affected by disease. Rat ELA was overexpressed in the heart via AAV9 vector by a single intravenous injection. ELA-treated animals showed delayed onset of blood pressure elevation. Prior to high-salt diet, a reduction in the fractional sodium and chloride excretion was observed in rats given the AAV9-ELA vector. After three months on a high-salt diet, ELA preserved glomerular architecture, decreased renal fibrosis and suppressed expression of fibrosis-associated genes in the kidneys. CONCLUSION ELA is constitutively expressed in renal collecting ducts in rats. Sustained AAV-ELA expression may offer a potential long-term therapy for hypertension and renal remodeling.

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen

Consequences of age on the effects of hyperbaric oxygen (HBO) on bone marrow (BM) derived stem cells and progenitors (SCPs) are largely unknown. We treated 2- and 18-month old C57BL/6 female mice by HBO. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-β and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold). Our data demonstrate the impact of aging on the response of BM SCPs to HBO and indicate the potentially different age-related benefit of HBO in wound healing and tissue remodeling.

Arterial Remodeling in B-Type Natriuretic Peptide Knock-Out Females.

Sexual dimorphisms are recognized in cardiovascular conditions such as hypertension, stroke, thrombosis and vasculitis. B-type natriuretic peptide (BNP) is a guanylyl cyclase A (GC-A) agonist. The anti-hypertensive, vasodilatory, anti-fibrotic, and anti-hypertrophic properties of BNP are well established in male animal models. Although circulating BNP levels are higher in women, when compared to age-matched men, the cardiovascular protective propensity of BNP in females is poorly understood. We assessed the cardiovascular consequences of BNP deletion in genetically null (Nppb−/−) female rat lines. Throughout the study, blood pressure (BP) remained uninfluenced by genotype, and cardiorenal consequences of BNP knock out remained minor. Unexpectedly, approximately 60% of Nppb−/− females developed mesenteric polyarteritis-nodosa (PAN)-like vasculitis in their life span, some as early as 4 months of age. Mesenteric lesions involved intense arterial remodeling, progressive inflammation, occluded lumens, and less frequently intestinal necrosis and multiple visceral arterial aneurysms. Cumulative pathologies resulted in a significant decline in survival of the Nppb−/− female. This study highlights BNP’s vasoprotective propensity, bringing to light a possible sex specific difference in the cardiovascular protection provided by BNP. Defects in the BNP/GC-A/cGMP pathway may play a role in arteriopathies in women, while GC-A agonists may provide effective therapy for arteritis.

307. Hitting the Kidney from All Angles: Using Direct Intrarenal Pelvis Injection Combined with Serotype and Promoter Targeting to Achieve Renal AAV Gene Delivery

Using adeno-associated virus (AAV)-based gene therapy to treat renal diseases is an underdeveloped area. Renal anatomy and physiology make gene delivery to this organ especially difficult. Multiple AAV serotypes and routes of entry in rodents have been tried, with the majority of combinations targeting the renal tubules. To improve upon the current methods of AAV renal transduction for future use in the clinical setting, we employed direct intrarenal pelvis injections of AAV2, 6, 8 and 9 with the luciferase (Luc) or GFP transgene under the control of the cytomegalovirus (CMV) or putative kidney-specific promoters from myo-inositol oxygenase(MIOX), acyl-CoA synthetase medium-chain family member 2 (Acsm2), uromodulin (UMOD), kidney androgen-regulated protein (Kap) and solute carrier family 34 (sodium phosphate), member 1 (Slc34a1) mouse genes in C57Bl/6J mice. Renal luciferase transgene expression was strongest with AAV6, 8 and 9-CMV-Luc. AAV6 and 8-CMV-Luc resulted in stronger expression in the injected kidney, whereas AAV9-CMV-Luc showed equal, bilateral renal expression. AAV9 with the UMOD promoter gave the strongest and most highly renal-specific luciferase expression among all serotype and kidney promoter combinations. Using the GFP transgene we further established AAV transduction of multiple cell types in the kidney. AAV8-CMV-GFP transduced both glomerular and tubular renal cells, while AAV9-CMV-GFP most strikingly transduced periglomerular tubule cells. Both AAV8 and AAV9-UMOD-GFP solely transduced renal tubule cells. We are currently testing AAV variants isolated from clinical urine samples to determine their ability to transduce renal cells in vivo. Our results demonstrate efficient renal transduction by direct intrarenal pelvis injection of AAV vectors. Coupling specific renal cell tropisms of AAV serotypes and renal cell-type-specific promoters, along with the intrarenal pelvis injection strategy would pave the way for a renal-targeted AV gene therapy.

8. Further Characterization of U2 snRNP Mediated Restriction of AAV Vector Transduction

Improving adeno-associated virus (AAV) vector transduction efficiency is central to the development of its continued, widespread use in gene therapy. More effective AAV transduction would reduce vector doses required for efficient gene delivery, minimizing the risks associated with high dose AAV vectors. Several treatments have been reported to increase AAV vector transduction, including adenoviral co-infection. Through screening of a siRNA library, we recently identified U2 snRNP as a host restriction factor for AAV vector transduction. Disruption of U2 snRNP spliceosome and associated proteins, including PHF5A, SF3B1, SF3B2 and U2AF1, potently enhanced AAV vector transduction. Relevant to gene therapy applications of AAV vectors, meayamycin B, a powerful SF3B1 inhibitor, allowed for substantial increases in AAV vector transduction (up to 400-fold). This post-entry restriction appeared to occur after the second-strand synthesis but before transgene expression or accumulation of transgene transcripts and independently of the cellular splicing machinery. No notable changes were found in the cytoplasmic trafficking, nuclear entry, or genome release of AAV vector infection by U2 snRNP inhibition. Here, we further studied the mechanism(s) underlying the U2 snRNP-mediated block of AAV vectors. We first tested another commercially available U2 snRNP inhibitor, pladienolide B (PladB) and verified that PladB treatment also showed substantial dose dependent increases (up to 700-fold) in AAV vector transduction. We next studied the relationship between adenoviral co-infection and U2 snRNP inhibition. Adenoviral co-infection alone enhanced AAV vector transduction up to 30-fold, while adenoviral co-infection, in addition to genetic or pharmacological U2 snRNP inhibition, showed marginal additive effects, suggesting a common pathway targeted by adenoviral co-infection and U2 snRNP inhibition in enhanced AAV vector infection. Using a series of plasmids providing adenoviral helper functions, we have identified Ad5 E4, but not the E2A and VA RNA genes, as partially responsible for the enhanced AAV transduction by adenoviral co-infection through U2 snRNP inhibition. Since the effects of U2 snRNP inhibition are most likely on the regulation of transgene expression, we also assessed the epigenetic modifications of the AAV2 genome in the presence or absence of PladB. Through chromosome immunoprecipitation (ChIP) assays we mapped histone recruitment patterns at different regions of the AAV vector epigenome. PladB treatment reproducibly enhanced recruitment of histone H2b and H3 proteins to the AAV CMV promoter, luciferase transgene, and polyA regions up to 3-fold. Our preliminary study also suggested that U2 snRNP inhibition altered specific histone modification patterns on the AAV vector genome. We are in the process of verifying the influence of U2 snRNP inhibition, as well as AAV capsid variations, on epigenetic modifications of the AAV vector epigenome. Better understanding the underlying mechanism would provide novel insights into host-virus interactions and could lead to the rational design of next generation AAV vectors with improved transduction efficiencies and safety profiles.