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Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03–mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9–mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches. Editor's summary: Mutations in the podocin-encoding NPHS2 gene can cause steroid-resistant nephrotic syndrome in children, with no effective targeted treatments available. Here, Ding and colleagues demonstrated the effectiveness of adeno-associated virus (AAV)-LK03 at transducing human podocytes in vitro. They then developed an AAV-based gene therapy to treat NPHS2 mutations using a minimal human nephrin promoter and tested it using AAV serotype 2/9, which can transduce murine podocytes, in two different mouse models of disease. Treatment before induction of disease and after disease onset reduced albuminuria in nephrotic mice, providing proof-of-concept that gene therapy might be a viable treatment strategy for patients with monogenic causes of nephrotic syndrome. —Melissa Norton [ABSTRACT FROM AUTHOR]

Background: Chronic gastroenteropathies, including gluten sensitivity and marmoset wasting syndrome, frequently occur in captive colonies of common marmosets (Callithrix jacchus). Early identification and diagnosis of affected animals are desirable. Endoscopic examination of the colon in marmosets is described, but the small intestine can harbor significant mucosal lesions not representing those in the colon. Evaluating the small intestine currently requires invasive surgical biopsies due to the small patient size, carrying a risk of severe complications. Methods: Endoscopic intubation and multisite biopsy of the duodenum/proximal jejunum are demonstrated in 10 marmosets under general anesthesia. Results: Esophagogastroduodenoscopy with colonoscopy efficiently aid in examining the gastrointestinal tract and obtaining an antemortem histologic diagnosis in marmosets with chronic gastrointestinal signs. Conclusions: This minimally invasive technique is feasible in marmosets. Future investigations into the pathogenesis of chronic gastroenteropathies will benefit from these data, leading to improved animal welfare and better individual and colony health management. [ABSTRACT FROM AUTHOR]

Introduction: The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half‐life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). Aim: Adeno‐associated viral vectors (AAV) mediating expression of hFIX‐Alb and hFIX‐Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half‐life translates to higher plasma levels of FIX. Methods: Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. Results: Both hFIX‐Alb and hFIX‐Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV‐mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX‐fusion proteins were comparable to wild‐type hFIX. However, their expression levels were threefold lower than wild‐type hFIX in vivo most likely due to inefficient secretion. Conclusion: This, the first, evaluation of hFIX‐fusion proteins in the context of AAV gene transfer suggests that the hFIX‐fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches. [ABSTRACT FROM AUTHOR]

Background Copolymers based on poly-[ N-(2-hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer-coating techniques can also be used to modify and retarget adeno-associated virus (AAV) types 5 and 8. Methods Three strategies for modifying transductional targeting of AAV were employed. The first involved direct reaction of AAV5 or AAV8 with amino-reactive HPMA copolymer. The second approach used carbodiimide (EDC) chemistry to increase the number of surface amino groups on the AAV5 capsid, thereby improving coating efficiency. In the third approach, the AAV5 genome was isolated from capsid proteins and delivered in a synthetic polyplex consisting of polyethylenimine (PEI) and HPMA. Results Efficient covalent attachment of HPMA copolymer to AAV5 could only be achieved following modification of the virus with EDC. Coating inhibited sialic acid dependent infection and provided a platform for retargeting via new ligands, including basic fibroblast growth factor. Retargeted infection was shown to be partially resistant to neutralising antisera. Delivery of AAV5 genomes using PEI and HPMA was efficient and provided absolute control of tropism and protection from antisera. In contrast AAV8 could be reacted directly with HPMA copolymer and allowed specific retargeting via the epidermal growth factor receptor, but gave no protection against neutralising antisera. Conclusions Reactive HPMA polymers can be used to ablate the natural tropism of both AAV8 and EDC-modified AAV5 and enable receptor-specific infection by incorporation of targeting ligands. These data show transductional targeting strategies can be used to improve the versatility of AAV vectors. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Vectors based on adeno-associated virus-8 (AAV8) have shown efficiency and efficacy for liver-directed gene therapy protocols following intravascular injection, particularly in relation to haemophilia gene therapy. AAV8 has also been proposed for gene therapy targeted at skeletal and cardiac muscle, again via intravascular injection. It is important to assess vector targeting at the level of virion accumulation and transgene expression in multiple species to ascertain potential issues relating to species variation in infectivity profiles. Methods: We used AAV8 vectors expressing human factor IX (FIX) from the liver-specific LP-1 promoter and administered this virus via the intravascular route of injection into 12 week old Wistar Kyoto rats. We assessed FIX levels in serum by ELISA and transgene expression at sacrifice by immunohistochemistry using anti-FIX antibodies. Vector DNA levels in organs we determined by real time PCR. Results: Administration of 1 × 1011 or 5 × 1011 scAAV8-LP1-hFIX vector particles/rat resulted in efficient production of physiological hFIX levels, respectively in blood assessed 4 weeks postinjection. This was maintained for the 4 month duration of the study. At 4 months we observed liver persistence of vector with minimal non-hepatic distribution. Conclusion: Our results demonstrate that AAV8 is a robust vector for delivering therapeutic genes into rat liver following intravascular injection. [ABSTRACT FROM AUTHOR]