Gene Therapy 2017: Progress and Future Directions

Keeler, Allison M.; ElMallah, Mai K.; Flotte, Terence R.

doi:10.1111/cts.12466

Cited by 121 publications

(71 citation statements)

References 118 publications

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“…The efficient editing, PCSK9 reduction, and diminished serum cholesterol indicate the successful delivery and activity of NmeCas9 at the Pcsk9 locus. 15 SpyCas9 delivered by viral vectors is known to elicit host immune responses (19,57). To investigate if the mice injected with AAV8-sgRNA-hNmeCas9 generate anti-NmeCas9 antibodies, we used sera from the treated animals to perform IgG1 ELISA.…”

Section: In Vivo Genome Editing By Nmecas9 Delivered By a Raav Vectormentioning

confidence: 99%

“…Compared to other viral vectors, rAAV persists in concatemeric, episomal forms, while eliciting mild immune responses (12)(13)(14). The usefulness of rAAV-based delivery for gene therapy is reflected in the number of clinical trials involving rAAV (15). One of the most exciting advancements for rAAV gene therapy field has been the FDA's recent market approval of a therapy for RPE65-mediated inherited retinal disease (IRD), the first of its kind in the United States (16).…”

Section: Introductionmentioning

confidence: 99%

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All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Ibraheim¹,

Song²,

Mir³

et al. 2018

Preprint

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Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. Here we demonstrate that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.3 SignificanceWe and others have shown that NmeCas9 is an efficient Cas9 nuclease with a high degree of specificity in cultured human cells. NmeCas9 has a unique PAM that expands the targeting range of Cas9-based genome editing. Since NmeCas9 is more compact than SpyCas9, its delivery with its guide-RNA in a single recombinant adeno-associated virus becomes a viable option. Furthermore, NmeCas9 has an unusually low off-target profile. These characteristics make NmeCas9 an ideal candidate for in vivo genome editing, including therapeutic gene knockouts, as we demonstrate in the mouse liver. We anticipate that successful in vivo delivery of this accurate and effective Cas9 can advance therapeutic editing in humans.

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Section: In Vivo Genome Editing By Nmecas9 Delivered By a Raav Vectormentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Ibraheim¹,

Song²,

Mir³

et al. 2018

Preprint

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“…New technologies in gene therapy such as stabilized therapeutic mRNAs and gene-editing methods such as CRISPR offer the promise of modifying disease genes or replacing them, temporarily if not permanently. Progress in gene therapy has been reviewed elsewhere and will not be covered here [89, 90]. In the interim, while the inborn errors community awaits the clinical availability of gene replacement therapies, other strategies continue to be pursued.…”

Section: Advances In Therapymentioning

confidence: 99%

Advances in the Understanding and Treatment of Mitochondrial Fatty Acid Oxidation Disorders

Goetzman

2017

Curr Genet Med Rep

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Purpose of review This review focuses on advances made in the past three years with regards to understanding the mitochondrial fatty acid oxidation (FAO) pathway, the pathophysiological ramifications of genetic lesions in FAO enzymes, and emerging therapies for FAO disorders. Recent findings FAO has now been recognized to play a key energetic role in pulmonary surfactant synthesis, T-cell differentiation and memory, and the response of the proximal tubule to kidney injury. Patients with FAO disorders may face defects in these cellular systems as they age. Aspirin, statins, and nutritional supplements modulate the rate of FAO under normal conditions and could be risk factors for triggering symptoms in patients with FAO disorders. Patients have been identified with mutations in the ACAD9 and ECHS1 genes, which may represent new FAO disorders. New interventions for long-chain FAODs are in clinical trials. Finally, post-translational modifications that regulate fatty acid oxidation protein activities have been characterized that represent important new therapeutic targets. Summary Recent research has led to a deeper understanding of FAO. New therapeutic avenues are being pursued that may ultimately cause a paradigm shift for patient care.

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“…Basic biology and the study of the function of the genome has been based on the availability of function deficient models, either cells or organisms, and the association of these loss-of-function with gene mutations. The availability of these models has allowed the research of human genetic diseases and, even, the development of gene therapy strategies for their treatment (Keeler, ElMallah, and Flotte 2017). The availability of endonucleases that can be directed to interact with very precise position in the cell genome has allowed the generation of knock-out models of any desired gene or position (Sakuma and Woltjen 2014; Silva et al 2011; Deng et al 2012).…”

Section: Introductionmentioning

confidence: 99%

In vitroandIn vivoGenetic Disease Modelling via NHEJ-precise deletions using CRISPR/Cas9

López-Manzaneda¹,

Ojeda-Pérez

Zabaleta

et al. 2020

Preprint

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49Development of advanced gene and cell therapies for the treatment of genetic diseases requires 50 confident animal and cellular models to test their efficacy and is crucial in the cases where no 51 primary samples from patients are available. CRISPR/Cas9 technology, has become one of the 52 most spread endonuclease tools for editing the genome at will. Moreover, it is known that the use 53 of two guides tends to produce the precise deletion between the guides via NHEJ. Different 54 distances between guides were tested (from 8 to 500 base pairs). We found that distances 55 between the two cutting sites and orientation of Cas9 protein-DNA interaction are important for 56 the efficiency within the optimal range (30-60 bp), we could obtain new genetically reproducible 57 models for two rare disease, a Pyruvate Kinase Deficiency model, using human primary cells, and a 58 (for in vivo primary hyperoxaluria therapeutic mouse model. We demonstrate that the use of a 2-59 guideRNAs at the optimal distance and orientation is a powerful strategy for disease modelling in 60 those diseases where the availability of primary cells is limited. 61 62 63 64 65 standard chow and water. Agxt1-/-mice were genotyped as described (Salido et al. 2006). Age-127 matched C57BL/6J mice (Harlan laboratories) were used as control animals. A final dose of 1x10 13 128 vg/kg (5x10 12 vg/kg/vector) was administered to 12-14-week-old Agxt1-/-male animals by 129 intravenous injection. Animals were sacrificed and livers harvested 21 days after the 130 administration of the vectors. 131 132 133

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Gene Therapy 2017: Progress and Future Directions

Cited by 121 publications

References 118 publications

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Advances in the Understanding and Treatment of Mitochondrial Fatty Acid Oxidation Disorders

In vitroandIn vivoGenetic Disease Modelling via NHEJ-precise deletions using CRISPR/Cas9

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