AAV-p40 Bioengineering Platform for Variant Selection Based on Transgene Expression

The power of AAV directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent selection platforms or PCR-based capsid DNA recovery methods, which can bias the selection towards efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform to existing replication competent strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection

Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver

Analysis of expression and prognosis of KLK7 in ovarian cancer

Ovarian cancer is one of the common malignant tumors in female reproductive organs. Kallikrein-related peptidase (KLK) 7 is a secreted serine peptidase that is related to different cancer. To investigate the expression and significance of KLK7 in ovarian cancer

AAV-delivered hepato-adrenal cooperativity in steroidogenesis: Implications for gene therapy for congenital adrenal hyperplasia

Despite the availability of life-saving corticosteroids for 70 years, treatment for adrenal insufficiency is not able to recapitulate physiological diurnal cortisol secretion and results in numerous complications. Gene therapy is an attractive possibility for monogenic adrenocortical disorders such as congenital adrenal hyperplasia; however, requires further development of gene transfer/editing technologies and knowledge of the target progenitor cell populations. Vectors based on adeno-associated virus are the leading system for direct in vivo gene delivery but have limitations in targeting replicating cell populations such as in the adrenal cortex. One strategy to overcome this technological limitation is to deliver the relevant adrenocortical gene to a currently targetable organ outside of the adrenal cortex. To explore this possibility, we developed a vector encoding human 21-hydroxylase and directed expression to the liver in a mouse model of congenital adrenal hyperplasia. This extra-adrenal expression resulted in reconstitution of the steroidogenic pathway. Aldosterone and renin levels normalized, and corticosterone levels improved sufficiently to reduce adrenal hyperplasia. This strategy could provide an alternative treatment option for monogenic adrenal disorders, particularly for mineralocorticoid defects. These findings also demonstrate, when targeting the adrenal gland, that inadvertent liver transduction should be precluded as it may confound data interpretation

Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids

Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic in vivo comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model

Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Abstract Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies