Browsing by Author "De Giorgi, Marco"

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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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    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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    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.