Targeting the Apoa1 locus for liver-directed gene therapy

Giorgi, M. De; Li, Ang; Hurley, Ayrea; Barzi, Mercedes; Doerfler, Alexandria M.; Cherayil, Nikitha; Smith, Harrison E.; Brown, J.; Lin, Charles Y.; Bissig, Karl Dimiter; Bao, Gang; Lagor, William R.

doi:10.1016/j.omtm.2021.04.011

Cited by 10 publications

(7 citation statements)

References 49 publications

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“…However, differences in recombination rate may be also affected by the targeting position in the albumin gene (1st intron in Wang et al, vs. exon 14 in our approach). Others have also used a similar approach in adult animals targeting the ApoA1 gene with AAV doses that were approximately 50× higher than the one in our study (De Giorgi et al, 2021).…”

Section: Discussionmentioning

confidence: 92%

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Lisjak

Caneva²,

Marais³

et al. 2022

Front. Genome Ed.

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Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

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Section: Discussionmentioning

confidence: 92%

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Lisjak

Caneva²,

Marais³

et al. 2022

Front. Genome Ed.

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“…73 Numerous gene insertion and gene editing approaches have corrected mouse models of this disease within a matter of weeks. [74][75][76][77] Despite the low degree of correction needed, residual liver cancer risk in tyrosinemia, and the availability of an approved drug nitisinone, has discouraged clinical development of AAV gene therapy. A selective advantage for corrected hepatocytes also exists for alpha1-antitrypsin deficiency, 78 Wilson disease, 79 methylmalonic academia, 80 and likely a few others.…”

Section: Liver Aav Gene Therapymentioning

confidence: 99%

“…127 Targeted transgene insertion is another popular approach which can be accomplished with promoterless AAV cassettes, leveraging regulatory elements of highly expressed genes in the liver such as albumin (ALB) 128 or apolipoprotein A1 (APOA1). 74 Targeting highly expressed loci with Zinc Fingers or CRISPR/Cas9 can dramatically increase the ratio of homology directed repair (HDR) events. 74,80,129,130 This formed the foundation for three clinical trials by Sangamo therapeutics for hemophilia B, Mucopolysaccharoidosis I and II (MPS I and II).…”

Section: Achieving Permanent Expressionmentioning

confidence: 99%

“…74 Targeting highly expressed loci with Zinc Fingers or CRISPR/Cas9 can dramatically increase the ratio of homology directed repair (HDR) events. 74,80,129,130 This formed the foundation for three clinical trials by Sangamo therapeutics for hemophilia B, Mucopolysaccharoidosis I and II (MPS I and II). 86 In these trials, AAV transgene cassettes were co-delivered with two other AAV vectors encoding Zinc Finger Nucleases (ZFN) for integration into intron 1 of the ALB gene.…”

Section: Achieving Permanent Expressionmentioning

confidence: 99%

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Liver directed adeno‐associated viral vectors to treat metabolic disease

Chuecos

Lagor

2023

J of Inher Metab Disea

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The liver is the metabolic center of the body and an ideal target for gene therapy of inherited metabolic disorders (IMDs). Adeno‐associated viral (AAV) vectors can deliver transgenes to the liver with high efficiency and specificity and a favorable safety profile. Recombinant AAV vectors contain only the transgene cassette, and their payload is converted to non‐integrating circular double‐stranded DNA episomes, which can provide stable expression from months to years. Insights from cellular studies and preclinical animal models have provided valuable information about AAV capsid serotypes with a high liver tropism. These vectors have been applied successfully in the clinic, particularly in trials for hemophilia, resulting in the first approved liver‐directed gene therapy. Lessons from ongoing clinical trials have identified key factors affecting efficacy and safety that were not readily apparent in animal models. Circumventing pre‐existing neutralizing antibodies to the AAV capsid, and mitigating adaptive immune responses to transduced cells are critical to achieving therapeutic benefit. Combining the high efficiency of AAV delivery with genome editing is a promising path to achieve more precise control of gene expression. The primary safety concern for liver gene therapy with AAV continues to be the small risk of tumorigenesis from rare vector integrations. Hepatotoxicity is a key consideration in the safety of neuromuscular gene therapies which are applied at substantially higher doses. The current knowledge base and toolkit for AAV is well developed, and poised to correct some of the most severe IMDs with liver‐directed gene therapy.

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“…A similar strategy to albumin locus insertion has also been described involving insertion of the therapeutic gene downstream of the apolipoprotein A1 (Apoa1) gene, which also has high expression in the liver. [ 61 ] Apoa1‐targeted FAH can correct and rescue mice suffering from HT1.…”

Section: Insertion Of the Therapeutic Genementioning

confidence: 99%

mRNA and gene editing: Late breaking therapies in liver diseases

Zabaleta

Torella

Weber³

et al. 2022

Hepatology

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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.

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Targeting the Apoa1 locus for liver-directed gene therapy

Cited by 10 publications

References 49 publications

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice

Liver directed adeno‐associated viral vectors to treat metabolic disease

mRNA and gene editing: Late breaking therapies in liver diseases

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