Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

Cross-species permissivity: structure of a goat adeno-associated virus and its complex with the human receptor, AAVR

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains Polycystic Kidney Disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA studies, it is shown that AAVGo.1 binds to human AAVR (huAAVR) more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of huAAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure, except for disordering of a neighboring surface loop. Only 4 of the 42 capsid protein sequence differences between AAVGo.1 and AAV5 occur at the PKD1 binding interface. These result in only minor conformational changes in AAVR, including a near rigid domain rotation with maximal displacement of the receptor by ~1 Å. A picture emerges of two classes of AAV with completely different modes of binding to the same AAVR receptor, but within each class atomic interactions are mostly conserved. IMPORTANCE Adeno-Associated Virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor huAAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

Cryo-EM Structure of Adeno-associated virus-4 at 2.2 Å resolution

Adeno-associated virus (AAV) is the vector of choice for several approved gene therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, we present a high-resolution cryo-electron microscopy structure (2.2 Å) for AAV serotype 4 (AAV4). The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, 12 and rh32.33) is unknown. Other AAVs interact with the same cell receptor, Adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like [PKD]-1 domain of AAVR while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR, provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear sufficient to exclude binding with PKD1.

Adeno-associated Virus Receptor-binding: Flexible Domains and Alternative Conformations through cryo-Electron Tomography of AAV2 and AAV5 complexes

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE: With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.

Gene-Based Therapeutics for Inherited Retinal Diseases

Inherited retinal diseases (IRDs) are a heterogenous group of orphan eye diseases that typically result from monogenic mutations and are considered attractive targets for gene-based therapeutics. Following the approval of an IRD gene replacement therapy for Leber’s congenital amaurosis due to RPE65 mutations, there has been an intensive international research effort to identify the optimal gene therapy approaches for a range of IRDs and many are now undergoing clinical trials. In this review we explore therapeutic challenges posed by IRDs and review current and future approaches that may be applicable to different subsets of IRD mutations. Emphasis is placed on five distinct approaches to gene-based therapy that have potential to treat the full spectrum of IRDs: 1) gene replacement using adeno-associated virus (AAV) and nonviral delivery vectors, 2) genome editing via the CRISPR/Cas9 system, 3) RNA editing by endogenous and exogenous ADAR, 4) mRNA targeting with antisense oligonucleotides for gene knockdown and splicing modification, and 5) optogenetic approaches that aim to replace the function of native retinal photoreceptors by engineering other retinal cell types to become capable of phototransduction.

TPRG1 contributes to inflammation and cell proliferation of cystitis glandularis through regulating NF-кB/COX2/PGE2 axis

Cystitis glandularis is characterized by chronic inflammation and hyperproliferation of bladder mucosa, and contributes to progression of bladder adenocarcinoma. TPRG1 (Tumor Protein P63 Regulated 1) is related to cellular inflammatory response, and dysregulation of TPRG1 in tumor tissues is associated with tumor early recurrence. The effect of TPRG1 on cystitis glandularis was investigated in this study. Firstly, bladder specimen were isolated from patients with cystitis glandularis and E. coli-induced cystitis rat. Expression of TPRG1 was found to be up-regulated in the bladder specimen. Moreover, adeno-associated virus (AAV)-mediated silence of TPRG1 was delivered into rat, and data from hematoxylin and eosin (H & E) staining showed that injection with AAV-shTPRG1 ameliorated E. coli-induced histological changes in bladder tissues of rats, and suppressed the inflammatory response. Secondly, TPRG1 was also increased in primary cystitis glandularis cells. Knockdown of TPRG1 decreased cell proliferation of primary cystitis glandularis cells, and suppressed the migration. Thirdly, cyclooxygenase-2 (COX-2) was up-regulated in the bladder specimen isolated from patients with cystitis glandularis and E. coli-induced cystitis rat. Injection with AAV-shTPRG1 reduced protein expression of COX-2, p65 and prostaglandin E2 (PGE2) in the bladder specimen. Lastly, interference of COX-2 attenuated TPRG1 over-expression-induced increase of cell proliferation and migration in the primary cystitis glandularis cells. In conclusion, TPRG1 promoted inflammation and cell proliferation of cystitis glandularis through activation of NF-кB/COX2/PGE2 axis.

Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Duchenne Muscular Dystrophy: The Issue of Transgene Persistence

Duchenne muscular dystrophy (DMD) is an X-linked recessive, infancy-onset neuromuscular disorder characterized by progressive muscle weakness and atrophy, leading to delay of motor milestones, loss of autonomous ambulation, respiratory failure, cardiomyopathy, and premature death. DMD originates from mutations in the DMD gene that result in a complete absence of dystrophin. Dystrophin is a cytoskeletal protein which belongs to the dystrophin-associated protein complex, involved in cellular signaling and myofiber membrane stabilization. To date, the few available therapeutic options are aimed at lessening disease progression, but persistent loss of muscle tissue and function and premature death are unavoidable. In this scenario, one of the most promising therapeutic strategies for DMD is represented by adeno-associated virus (AAV)-mediated gene therapy. DMD gene therapy relies on the administration of exogenous micro-dystrophin, a miniature version of the dystrophin gene lacking unnecessary domains and encoding a truncated, but functional, dystrophin protein. Limited transgene persistence represents one of the most significant issues that jeopardize the translatability of DMD gene replacement strategies from the bench to the bedside. Here, we critically review preclinical and clinical studies of AAV-mediated gene therapy in DMD, focusing on long-term transgene persistence in transduced tissues, which can deeply affect effectiveness and sustainability of gene replacement in DMD. We also discuss the role played by the overactivation of the immune host system in limiting long-term expression of genetic material. In this perspective, further studies aimed at better elucidating the need for immune suppression in AAV-treated subjects are warranted in order to allow for life-long therapy in DMD patients.