Abstract
Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered nucleases induces a double-stranded break (DSB) in the target genomic locus, thereby activating DNA repair mechanisms. DSBs triggered by nucleases are repaired either by the nonhomologous end-joining or the homology-directed repair pathway, enabling efficient gene editing. While there are several ongoing ex vivo genome editing clinical trials, current research underscores the therapeutic potential of CRISPR/Cas-based (clustered regularly interspaced short palindrome repeats-associated Cas nuclease) in vivo gene editing. In this review, we provide an overview of the CRISPR/Cas-mediated in vivo genome therapy applications and explore their prospective clinical translatability to treat human monogenic disorders. In addition, we discuss the various challenges associated with in vivo genome editing technologies and strategies used to circumvent them. Despite the robust and precise nuclease-mediated gene editing, a promoterless, nuclease-independent gene targeting strategy has been utilized to evade the drawbacks of the nuclease-dependent system, such as off-target effects, immunogenicity, and cytotoxicity. Thus, the rapidly evolving paradigm of gene editing technologies will continue to foster the progress of gene therapy applications.
Highlights
GENE EDITING HAS emerged as one of the most revolutionary breakthroughs in the field of biomedical sciences over the past decade
We focus on the nuclease-dependent (CRISPR/Cas) HDRbased editing in vivo to treat human monogenic diseases, briefly evaluate the hurdles and mitigation strategies coupled with in vivo delivery, and discuss nuclease-free editing as an alternate gene targeting approach
The CRISPR/Cas toolbox has been used for gene regulation, epigenetic modification, drug development, and precision medicine providing personalized therapies based on specific targets and diagnostics, extensively reviewed elsewhere.[226,227]
Summary
GENE EDITING HAS emerged as one of the most revolutionary breakthroughs in the field of biomedical sciences over the past decade. CRISPR/Cas9-mediated editing and NHEJ successfully impaired mutant AAT and effectively ameliorated liver fibrosis in a humanized mouse model, supporting a potential therapeutic possibility of treating AATD patients.[55] An additional study utilized coinjection of a dual adeno-associated vector (AAV): one encoding Cas[9] and another expressing an AAT gRNA and an HDR donor template into the liver of a transgenic mouse model This approach enabled precise AAT gene correction in vivo and partially restored. AATD, alpha-1 antitrypsin deficiency; AAV, Adeno-associated vector; ABE, adenine base editing; Ad, adenovirus; ALS, amyotrophic lateral sclerosis; CAR, chimeric antigen receptor; CRISPR/Cas, clustered regularly interspaced short palindrome repeats-associated Cas nuclease; dCas[9], dead Cas[9]; DMD, Duchenne muscular dystrophy; gRNA, guide RNA; HBV, hepatitis B virus; HDR, homology-directed repair; HIV, human immunodeficiency virus; HITI, homologyindependent targeted integration; HR, homologous recombination; HTI, hereditary tyrosinemia; LCA, Leber’s congenital amaurosis; LNP, lipid nanoparticles; NHEJ, nonhomologous end-joining; PNA, peptide nucleic acids; RNP, ribonucleoprotein; SCD, sickle cell disease; sgRNA, single-guide RNA; TALEN, transcription activator-like effector nuclease; ZFN, zinc-finger nuclease. Polymer gel-based plasmid delivery enabling ZFN-based deletion of E7 oncogene in HPV16 and HPV18
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