Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights.

Today, adeno-associated virus (AAV)-based vectors are arguably the most promising in vivo gene delivery vehicles for durable therapeutic gene expression. Advances in molecular engineering, high-throughput screening platforms, and computational techniques have resulted in a toolbox of capsid variants with enhanced performance over parental serotypes. Despite their considerable promise and emerging clinical success, there are still obstacles hindering their broader use, including limited transduction capabilities, tissue/cell type-specific tropism and penetration into tissues through anatomical barriers, off-target tissue biodistribution, intracellular degradation, immune recognition, and a lack of translatability from preclinical models to clinical settings. Here, we first describe the transduction mechanisms of natural AAV serotypes and explore the current understanding of the systemic and cellular hurdles to efficient transduction. We then outline progress in developing designer AAV capsid variants, highlighting the seminal discoveries of variants which can transduce the central nervous system upon systemic administration, and, to a lesser extent, discuss the targeting of the peripheral nervous system, eye, ear, lung, liver, heart, and skeletal muscle, emphasizing their tissue and cell specificity and translational promise. In particular, we dive deeper into the molecular mechanisms behind their enhanced properties, with a focus on their engagement with host cell receptors previously inaccessible to natural AAV serotypes. Finally, we summarize the main findings of our review and discuss future directions.

Identification of Genes and Pathways Regulated by Lamin A in Heart.

Background Mutations in the LMNA gene, encoding LMNA (lamin A/C), causes distinct disorders, including dilated cardiomyopathies, collectively referred to as laminopathies. The genes (coding and noncoding) and regulatory pathways controlled by LMNA in the heart are not completely defined. Methods and Results We analyzed cardiac transcriptome from wild-type, loss-of-function (Lmna-/-), and gain-of-function (Lmna-/- injected with adeno-associated virus serotype 9 expressing LMNA) mice with normal cardiac function. Deletion of Lmna (Lmna-/-) led to differential expression of 2193 coding and 629 long noncoding RNA genes in the heart (q<0.05). Re-expression of LMNA in the Lmna-/- mouse heart, completely rescued 501 coding and 208 non-coding and partially rescued 1862 coding and 607 lncRNA genes. Pathway analysis of differentially expressed genes predicted activation of transcriptional regulators lysine-specific demethylase 5A, lysine-specific demethylase 5B, tumor protein 53, and suppression of retinoblastoma 1, paired-like homeodomain 2, and melanocyte-inducing transcription factor, which were completely or partially rescued upon reexpression of LMNA. Furthermore, lysine-specific demethylase 5A and 5B protein levels were increased in the Lmna-/- hearts and were partially rescued upon LMNA reexpression. Analysis of biological function for rescued genes identified activation of tumor necrosis factor-α, epithelial to mesenchymal transition, and suppression of the oxidative phosphorylation pathway upon Lmna deletion and their restoration upon LMNA reintroduction in the heart. Restoration of the gene expression and transcriptional regulators in the heart was associated with improved cardiac function and increased survival of the Lmna-/- mice. Conclusions The findings identify LMNA-regulated cardiac genes and their upstream transcriptional regulators in the heart and implicate lysine-specific demethylase 5A and B as epigenetic regulators of a subset of the dysregulated genes in laminopathies.

Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids.

Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction.