R-3 Repository :: Browsing by Author "Li, Ang"

Browsing by Author "Li, Ang"

Now showing 1 - 8 of 8

Development and Characterization of Viral-Based Gene Editing In Vivo
(2020-08-12) Li, Ang; Bao, Gang
Adeno-Associated Viral (AAV) vectors packaging the CRISPR/Cas9 system (AAVCRISPR) 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 editing applications, permanent expression of non-human proteins such as the bacterially-derived Cas9 nuclease is undesirable. Recent studies indicate a high prevalence of neutralizing antibodies and T-cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. Additionally, persistent expression of CRISPR/Cas9 has the potential to increase the chances of off-target cutting. There is a need for efficient genome editing in vivo, with controlled transient expression of CRISPR/Cas9. The topic of my thesis covers the development of a self-deleting AAVCRISPR system that introduces insertion and deletion mutations into AAV episomes, understanding the effects AAV-CRISPR editing in vivo in a Cas9 immunized mouse model, and the characterization of AAV integrations into the genome in the context of CRISPR-based gene editing.
Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: A Proof-of-Concept Investigation
(Cell Press, 2020) Asthana, Vishwaratn; Stern, Brett S.; Tang, Yuqi; Bugga, Pallavi; Li, Ang; Ferguson, Adam; Asthana, Anantratn; Bao, Gang; Drezek, Rebekah A.; Bioengineering
Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment, including differentiating self from nonself, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein a new class of programmable self-assembling double-stranded RNA (dsRNA)-based cancer therapeutics that uniquely targets aberrant genetic sequences and in a functionally decoupled manner, undergoes oncogenic RNA-activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof of concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing sarcoma cells that are end blocked with phosphorothioate bonds and additionally sealed with a 2′-deoxyuridine (2′-U)-modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence but not wild type.
A GPU-based preconditioned Newton-Krylov solver for flexible multibody dynamics
(Wiley, 2015) Serban, Radu; Melanz, Daniel; Li, Ang; Stanciulescu, Ilinca; Jayakumar, Paramsothy; Negrut, Dan
This paper describes an approach to numerically approximate the time evolution of multibody systems with flexible (compliant) components. Its salient attribute is that at each time step, both the formulation of the system equations of motion and their numerical solution are carried out using parallel computing on graphics processing unit cards. The equations of motion are obtained using the absolute nodal coordinate formulation, yet any other multibody dynamics formalism would fit equally well the overall solution strategy outlined herein. The implicit numerical integration method adopted relies on a Newton-Krylov methodology and a parallel direct sparse solver to precondition the underlying linear system. The proposed approach, implemented in a software infrastructure available under an open-source BSD-3 license, leads to improvements in overall simulation times of up to one order of magnitude when compared with matrix-free parallel solution approaches that do not use preconditioning.
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.
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.
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.
Surface terminations and layer-resolved tunneling spectroscopy of the 122 iron pnictide superconductors
(American Physical Society, 2019) Li, Ang; Yin, Jia-Xin; Wang, Jihui; Wu, Zheng; Ma, Jihua; Sefat, Athena S.; Sales, Brian C.; Mandrus, David G.; McGuire, Michael A.; Jin, Rongying; Zhang, Chenglin; Dai, Pengcheng; Lv, Bing; Chu, Ching-Wu; Liang, Xuejin; Hor, P.-H.; Ting, C.-S.; Pan, Shuheng H.
The surface terminations of 122-type alkaline earth metal iron pnictides AEFe2As2(AE=Ca,Ba) are investigated with scanning tunneling microscopy/spectroscopy. Cleaving these crystals at a cryogenic temperature yields a large majority of terminations with an atomically resolved (√2 × √2)R45 or 1 × 2 lattice, as well as a very rare termination of 1 × 1 lattice symmetry. By analyzing the lattice registration and selective chemical marking, we identify these terminations as (√2 × √2)R45-reconstructed AE, 1 × 2-reconstructed As, and (√2 × √2)R45-reconstructed Fe surface layers, respectively. Layer-resolved tunneling spectroscopy on these terminating surfaces reveals a well-defined superconducting energy gap on the As terminations, while the gap features become weaker on the AE terminations and absent on the Fe terminations. The superconducting gap is hardly affected locally by the As or AE surface reconstructions. The definitive identification of the surface terminations and the associated spectroscopic signatures shed light on the essential roles of As and the pnictogen-iron-pnictogen trilayer building block in iron-based superconductivity.
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.

Browsing by Author "Li, Ang"

Results Per Page

Sort Options