Browsing by Author "Lagor, William R."

Now showing 1 - 7 of 7
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    In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia
    (American Heart Association, 2018) Pan, Xiaolu; Philippen, Leonne; Lahiri, Satadru K.; Lee, Ciaran; Park, So Hyun; Word, Tarah A.; Li, Na; Jarrett, Kelsey E.; Gupta, Rajat; Reynolds, Julia O.; Lin, Jean; Bao, Gang; Lagor, William R.; Wehrens, Xander H.T.; Bioengineering
    Rationale:Autosomal-dominant mutations in ryanodine receptor type 2 (RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.Objective:To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).Methods and Results:Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 (Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.Conclusions:AAV serotype 9–based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.
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    In vivo genome editing at the albumin locus to treat methylmalonic acidemia
    (Elsevier, 2021) Schneller, Jessica L.; Lee, Ciaran M.; Venturoni, Leah E.; Chandler, Randy J.; Li, Ang; Myung, Sangho; Cradick, Thomas J.; Hurley, Ayrea E.; Lagor, William R.; Bao, Gang; Venditti, Charles P.; Bioengineering
    Methylmalonic acidemia (MMA) is a metabolic disorder most commonly caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Although adeno-associated viral (AAV) gene therapy has been effective at correcting the disease phenotype in MMA mouse models, clinical translation may be impaired by loss of episomal transgene expression and magnified by the need to treat patients early in life. To achieve permanent correction, we developed a dual AAV strategy to express a codon-optimized MMUT transgene from Alb and tested various CRISPR-Cas9 genome-editing vectors in newly developed knockin mouse models of MMA. For one target site in intron 1 of Alb, we designed rescue cassettes expressing MMUT behind a 2A-peptide or an internal ribosomal entry site sequence. A second guide RNA targeted the initiator codon, and the donor cassette encompassed the proximal albumin promoter in the 5′ homology arm. Although all editing approaches were therapeutic, targeting the start codon of albumin allowed the use of a donor cassette that also functioned as an episome and after homologous recombination, even without the expression of Cas9, as an integrant. Targeting the albumin locus using these strategies would be effective for other metabolic disorders where early treatment and permanent long-term correction are needed.
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    LPA disruption with AAV-CRISPR potently lowers plasma apo(a) in transgenic mouse model: A proof-of-concept study
    (Elsevier, 2022) Doerfler, Alexandria M.; Park, So Hyun; Assini, Julia M.; Youssef, Amer; Saxena, Lavanya; Yaseen, Adam B.; De Giorgi, Marco; Chuecos, Marcel; Hurley, Ayrea E.; Li, Ang; Marcovina, Santica M.; Bao, Gang; Boffa, Michael B.; Koschinsky, Marlys L.; Lagor, William R.; Bioengineering
    Lipoprotein(a) (Lp(a)) represents a unique subclass of circulating lipoprotein particles and consists of an apolipoprotein(a) (apo(a)) molecule covalently bound to apolipoprotein B-100. The metabolism of Lp(a) particles is distinct from that of low-density lipoprotein (LDL) cholesterol, and currently approved lipid-lowering drugs do not provide substantial reductions in Lp(a), a causal risk factor for cardiovascular disease. Somatic genome editing has the potential to be a one-time therapy for individuals with extremely high Lp(a). We generated an LPA transgenic mouse model expressing apo(a) of physiologically relevant size. Adeno-associated virus (AAV) vector delivery of CRISPR-Cas9 was used to disrupt the LPA transgene in the liver. AAV-CRISPR nearly completely eliminated apo(a) from the circulation within a week. We performed genome-wide off-target assays to determine the specificity of CRISPR-Cas9 editing within the context of the human genome. Interestingly, we identified intrachromosomal rearrangements within the LPA cDNA in the transgenic mice as well as in the LPA gene in HEK293T cells, due to the repetitive sequences within LPA itself and neighboring pseudogenes. This proof-of-concept study establishes the feasibility of using CRISPR-Cas9 to disrupt LPA in vivo, and highlights the importance of examining the diverse consequences of CRISPR cutting within repetitive loci and in the genome globally.
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    The NIH Somatic Cell Genome Editing program
    (Springer Nature, 2021) Saha, Krishanu; Sontheimer, Erik J.; Brooks, P.J.; Dwinell, Melinda R.; Gersbach, Charles A.; Liu, David R.; Murray, Stephen A.; Tsai, Shengdar Q.; Wilson, Ross C.; Anderson, Daniel G.; Asokan, Aravind; Banfield, Jillian F.; Bankiewicz, Krystof S.; Bao, Gang; Bulte, Jeff W.M.; Bursac, Nenad; Campbell, Jarryd M.; Carlson, Daniel F.; Chaikof, Elliot L.; Chen, Zheng-Yi; Cheng, R. Holland; Clark, Karl J.; Curiel, David T.; Dahlman, James E.; Deverman, Benjamin E.; Dickinson, Mary E.; Doudna, Jennifer A.; Ekker, Stephen C.; Emborg, Marina E.; Feng, Guoping; Freedman, Benjamin S.; Gamm, David M.; Gao, Guangping; Ghiran, Ionita C.; Glazer, Peter M.; Gong, Shaoqin; Heaney, Jason D.; Hennebold, Jon D.; Hinson, John T.; Khvorova, Anastasia; Kiani, Samira; Lagor, William R.; Lam, Kit S.; Leong, Kam W.; Levine, Jon E.; Lewis, Jennifer A.; Lutz, Cathleen M.; Ly, Danith H.; Maragh, Samantha; McCray, Paul B.; McDevitt, Todd C.; Mirochnitchenko, Oleg; Morizane, Ryuji; Murthy, Niren; Prather, Randall S.; Ronald, John A.; Roy, Subhojit; Roy, Sushmita; Sabbisetti, Venkata; Saltzman, W. Mark; Santangelo, Philip J.; Segal, David J.; Shimoyama, Mary; Skala, Melissa C.; Tarantal, Alice F.; Tilton, John C.; Truskey, George A.; Vandsburger, Moriel; Watts, Jonathan K.; Wells, Kevin D.; Wolfe, Scot A.; Xu, Qiaobing; Xue, Wen; Yi, Guohua; Zhou, Jiangbing; Bioengineering
    The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium’s plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled—along with validated datasets—into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit—and the knowledge generated by its applications—as a means to accelerate the clinical development of new therapies for a wide range of conditions.
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    A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
    (Elsevier, 2019) Li, Ang; Lee, Ciaran M.; Hurley, Ayrea E.; Jarrett, Kelsey E.; De Giorgi, Marco; Lu, Weiqi; Balderrama, Karol S.; Doerfler, Alexandria M.; Deshmukh, Harshavardhan; Ray, Anirban; Bao, Gang; Lagor, William R.; Bioengineering
    Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.
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    Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease
    (Springer Nature, 2017) Jarrett, Kelsey E.; Lee, Ciaran M.; Yeh, Yi-Hsien; Hsu, Rachel H.; Gupta, Rajat; Zhang, Min; Rodriguez, Perla J.; Lee, Chang Seok; Gillard, Baiba K.; Bissig, Karl-Dimiter; Pownall, Henry J.; Martin, James F.; Bao, Gang; Lagor, William R.; Bioengineering
    Germline manipulation using CRISPR/Cas9 genome editing has dramatically accelerated the generation of new mouse models. Nonetheless, many metabolic disease models still depend upon laborious germline targeting, and are further complicated by the need to avoid developmental phenotypes. We sought to address these experimental limitations by generating somatic mutations in the adult liver using CRISPR/Cas9, as a new strategy to model metabolic disorders. As proof-of-principle, we targeted the low-density lipoprotein receptor (Ldlr), which when deleted, leads to severe hypercholesterolemia and atherosclerosis. Here we show that hepatic disruption of Ldlr with AAV-CRISPR results in severe hypercholesterolemia and atherosclerosis. We further demonstrate that co-disruption of Apob, whose germline loss is embryonically lethal, completely prevented disease through compensatory inhibition of hepatic LDL production. This new concept of metabolic disease modeling by somatic genome editing could be applied to many other systemic as well as liver-restricted disorders which are difficult to study by germline manipulation.
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    Targeting the Apoa1 locus for liver-directed gene therapy
    (Cell Press, 2021) De Giorgi, Marco; Li, Ang; Hurley, Ayrea; Barzi, Mercedes; Doerfler, Alexandria M.; Cherayil, Nikitha A.; Smith, Harrison E.; Brown, Jonathan D.; Lin, Charles Y.; Bissig, Karl-Dimiter; Bao, Gang; Lagor, William R.; Bioengineering
    Clinical application of somatic genome editing requires therapeutics that are generalizable to a broad range of patients. Targeted insertion of promoterless transgenes can ensure that edits are permanent and broadly applicable while minimizing risks of off-target integration. In the liver, the Albumin (Alb) locus is currently the only well-characterized site for promoterless transgene insertion. Here, we target the Apoa1 locus with adeno-associated viral (AAV) delivery of CRISPR-Cas9 and achieve rates of 6% to 16% of targeted hepatocytes, with no evidence of toxicity. We further show that the endogenous Apoa1 promoter can drive robust and sustained expression of therapeutic proteins, such as apolipoprotein E (APOE), dramatically reducing plasma lipids in a model of hypercholesterolemia. Finally, we demonstrate that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can correct and rescue the severe metabolic liver disease hereditary tyrosinemia type I. In summary, we identify and validate Apoa1 as a novel integration site that supports durable transgene expression in the liver for gene therapy applications.