mRNA and gene editing: Late breaking therapies in liver diseases.

The efficient delivery of RNA molecules to restore the expression of a missing or inadequately functioning protein in a target cell and the intentional specific modification of the host genome using engineered nucleases represent therapeutic concepts that are revolutionizing modern medicine. The initiation of several clinical trials using these approaches to treat metabolic liver disorders as well as the recently reported remarkable results obtained by patients with transthyretin amyloidosis highlight the advances in this field and show the potential of these therapies to treat these diseases safely and efficaciously. These advances have been possible due, firstly, to significant improvements made in RNA chemistry that increase its stability and prevent activation of the innate immune response and, secondly, to the development of very efficient liver-targeted RNA delivery systems. In parallel, the breakout of CRISPR/CRISPR-associated 9-based technology in the gene editing field has marked a turning point in in vivo modification of the cellular genome with therapeutic purposes, which can be based on gene supplementation, correction, or silencing. In the coming years we are likely to witness the therapeutic potential of these two strategies both separately and in combination. In this review we summarize the preclinical data obtained in animal models treated with mRNA as a therapeutic agent and discuss the different gene editing strategies applied to the treatment of liver diseases, highlighting both their therapeutic efficacy as well as safety concerns.

AdrA as a Potential Immunomodulatory Candidate for STING-Mediated Antiviral Therapy That Required Both Type I IFN and TNF-α Production.

Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrA was addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrA wt-treated mice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-αR (IFNAR)-/- animals were additionally treated with anti-TNF-α (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR-/- and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-α-treated IFNAR-/- animals. No effect of AdrA wt was seen in STING-deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-α are both required and act synergistically.

Durable immunogenicity, adaptation to emerging variants, and low-dose efficacy of an AAV-based COVID-19 vaccine platform in macaques.

The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

GPR108 Is a Highly Conserved AAV Entry Factor.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

Successful Repeated Hepatic Gene Delivery in Mice and Non-human Primates Achieved by Sequential Administration of AAV5ch and AAV1.

In the gene therapy field, re-administration of adeno-associated virus (AAV) is an important topic because a decrease in therapeutic protein expression might occur over time. However, an efficient re-administration with the same AAV serotype is impossible due to serotype-specific, anti-AAV neutralizing antibodies (NABs) that are produced after initial AAV treatment. To address this issue, we explored the feasibility of using chimeric AAV serotype 5 (AAV5ch) and AAV1 for repeated liver-targeted gene delivery. To develop a relevant model, we immunized animals with a high dose of AAV5ch-human secreted embryonic alkaline phosphatase (hSEAP) that generates high levels of anti-AAV5ch NAB. Secondary liver transduction with the same dose of AAV1-human factor IX (hFIX) in the presence of high levels of anti-AAV5ch NAB proved to be successful because expression/activity of both reporter transgenes was observed. This is the first time that two different transgenes are shown to be produced by non-human primate (NHP) liver after sequential administration of clinically relevant doses of both AAV5ch and AAV1. The levels of transgene proteins achieved after delivery with AAV5ch and AAV1 illustrate the possibility of both serotypes for liver targeting. Furthermore, transgene DNA and RNA biodistribution patterns provided insight into the potential cause of decrease or loss of transgene protein expression over time in NHPs.

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

Gene therapy for liver diseases - progress and challenges.

Gene therapy is poised to revolutionize modern medicine, with seemingly unlimited potential for treating and curing genetic disorders. For otherwise incurable indications, including most inherited metabolic liver disorders, gene therapy provides a realistic therapeutic option. In this Review, we discuss gene supplementation and gene editing involving the use of recombinant adeno-associated virus (rAAV) vectors for the treatment of inherited liver diseases, including updates on several ongoing clinical trials that are producing promising results. Clinical testing has been essential in highlighting many key translational challenges associated with this transformative therapy. In particular, the interaction of a patient's immune system with the vector raises issues of safety and the duration of treatment efficacy. Furthermore, several serious adverse events after the administration of high doses of rAAVs suggest greater involvement of innate immune responses and pre-existing hepatic conditions than initially anticipated. Finally, permanent modification of the host genome associated with rAAV genome integration and gene editing raises concerns about the risk of oncogenicity that require careful evaluation. We summarize the main progress, challenges and pathways forward for gene therapy for liver diseases.

Correlating physicochemical and biological properties to define critical quality attributes of a rAAV vaccine candidate.

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vector's physicochemical properties and biological potency. To this end, we evaluated an rAAV-based coronavirus disease 2019 (COVID-19) vaccine candidate that encodes the Spike antigen (AC3) and is produced by a commercially viable process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3, including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates, and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented, and data were correlated with the physicochemical analyses on thermally stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and, importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccine candidates as well as specifically for the rAAV vaccine application under study.

Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice.

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies-an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9-to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

Immunogenicity of an AAV-Based COVID-19 Vaccine in Murine Models of Obesity and Aging.

The SARS-CoV-2 pandemic has had a disastrous impact on global health. Although some vaccine candidates have been effective in combating SARS-CoV-2, logistical, economical, and sociological aspects still limit vaccine access globally. Recently, we reported on two room-temperature stable AAV-based COVID-19 vaccines that induced potent and protective immunogenicity following a single injection in murine and primate models. Obesity and old age are associated with increased mortality in COVID-19, as well as reduced immunogenicity and efficacy of vaccines. Here, we investigated the effectiveness of the AAVCOVID vaccine candidates in murine models of obesity and aging. Results demonstrate that obesity did not significantly alter the immunogenicity of either vaccine candidate. In aged mice, vaccine immunogenicity was impaired. These results suggest that AAV-based vaccines may have limitations in older populations and may be equally applicable in obese and non-obese populations.

Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Computed tomography and [18F]-FDG PET imaging provide additional readouts for COVID-19 pathogenesis and therapies evaluation in non-human primates.

Non-human primates (NHPs) are particularly relevant as preclinical models for SARS-CoV-2 infection and nuclear imaging may represent a valuable tool for monitoring infection in this species. We investigated the benefit of computed X-ray tomography (CT) and [18F]-FDG positron emission tomography (PET) to monitor the early phase of the disease in a large cohort (n = 76) of SARS-CoV-2 infected macaques. Following infection, animals showed mild COVID-19 symptoms including typical lung lesions. CT scores at the acute phase reflect the heterogeneity of lung burden following infection. Moreover, [18F]-FDG PET revealed that FDG uptake was significantly higher in the lungs, nasal cavities, lung-draining lymph nodes, and spleen of NHPs by 5 days postinfection compared to pre-infection levels, indicating early local inflammation. The comparison of CT and PET data from previous COVID-19 treatments or vaccines we tested in NHP, to this large cohort of untreated animals demonstrated the value of in vivo imaging in preclinical trials.

Novel vectors and approaches for gene therapy in liver diseases.

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates.

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

An AAV-based, room-temperature-stable, single-dose COVID-19 vaccine provides durable immunogenicity and protection in non-human primates.

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.

Inhibition of MicroRNA 6937 Delays Photoreceptor and Vision Loss in a Mouse Model of Retinitis Pigmentosa.

Inherited retinal dystrophies (IRDs) are a group of rare retinal conditions, including retinitis pigmentosa (RP), caused by monogenic mutations in 1 out of more than 250 genes. Despite recent advancements in gene therapy, there is still a lack of an effective treatment for this group of retinal conditions. MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that inhibit gene expression. Control of miRNAs-mediated protein expression has been described as a widely used mechanism for post-transcriptional regulation in many physiological and pathological processes in different organs, including the retina. Our main purpose was to test the hypothesis that modulation of a group of miRNAs can protect photoreceptor cells from death in the rd10 mouse model of retinitis pigmentosa. For this, we incorporated modulators of three miRNAs in adeno-associated viruses (AAVs), which were administered through sub-retinal injections. The results obtained indicate that inhibition of the miR-6937-5p slows down the visual deterioration of rd10 mice, reflected by an increased electroretinogram (ERG) wave response under scotopic conditions and significant preservation of the outer nuclear layer thickness. This work contributes to broadening our knowledge on the molecular mechanisms underlying retinitis pigmentosa and supports the development of novel therapeutic approaches for RP based on miRNA modulation.

In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9.

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30-60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.

Genetic-Based Approaches to Inherited Metabolic Liver Diseases.

In vertebrates, the liver is the central metabolic organ of the body, which carries out an estimated 500 functions that range from general detoxification to protein synthesis, bile production, metabolism of fats, carbohydrates, proteins, bilirubin, vitamin and mineral storage and it even has an immune function. Hepatocytes are considered the professional liver cells, which carry out all of these functions. With such a variety of tasks to perform, it is not surprising that more than 400 rare monogenic disorders of hepatic origin have been described. For many of these, liver transplantation remains the only curative strategy, however, this is limited by organ availability and requires lifelong immune suppression. The fact that liver transplantation is curative led to the assumption that the restoration of the expression of the defective gene would result in the resolution of the disease. Indeed, liver-directed gene therapy trials for hemophilia A and B have demonstrated the potential of gene therapy to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Thus, liver-directed gene therapy and gene editing strategies have emerged as promising alternatives to transplantation in inherited monogenic liver disorders. Herein, we review the advances and limitations of gene therapy for such disorders, covering therapeutic strategies based on gene addition and gene editing and the exciting clinical results obtained with the use of ribonucleic acid as therapeutic molecules.

Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates.

Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.