Biology subjects

Tsai, S. Q.

Publications and source records attributed to Tsai, S. Q..

11 recordsLinked to original sources

bioRxiv2024

In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor

Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (ACTA2) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.

bioengineering↗

bioRxiv2024

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease

Sickle cell disease (SCD) is a common severe blood disorder, caused by one major point mutation in the HBB gene. Current pharmacotherapies are only partially effective and potentially curative allogeneic hematopoietic stem cell transplantation (HSCT) is associated with immune toxicities. Genome editing of autologous patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers a potentially curative approach to treat SCD and circumvents some problems associated with allogeneic HSCT. Although the FDA has released guidelines for evaluating genome editing risks, it remains unclear how to best to assess the preclinical safety and efficacy of genome-edited cellular drug products to prepare for a clinical trial. Here we describe rigorous pre-clinical characterization and optimization of a therapeutic {gamma}-globin gene promoter editing strategy that supported an investigational new drug (IND) application cleared by the FDA. We compared targets in the {gamma}-globin promoter and BCL11A erythroid-specific enhancer, identified a lead candidate that potently induces HbF, and tested our approach in mobilized CD34+ HSPCs from normal donors and individuals with SCD. We observed efficient editing, induction of HbF to levels predicted to be therapeutic, and reduction of sickling in red blood cells derived from edited HSPCs. With single-cell western and RNA-seq analyses, we defined the heterogeneity and specificity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased genome-wide off-target discovery followed by multiplexed targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new discoveries on ex vivo genome editing of HSCs towards clinical trials for treating SCD and other blood disorders.

genomics↗

bioRxiv2024

CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of base editor genome-wide activity

Base editors (BE) enable programmable conversion of nucleotides in genomic DNA without double-stranded breaks and have substantial promise to become new transformative genome editing medicines. Sensitive and unbiased detection of base editor off-target effects is important for identifying safety risks unique to base editors and translation to human therapeutics, as well as accurate use in life sciences research. However, current methods for understanding the global activities of base editors have limitations in terms of sensitivity or bias. Here we present CHANGE-seq-BE, a novel method to directly assess the off-target profile of base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE is based on the principle of selective sequencing of adenine base editor modified genomic DNA in vitro, and provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of base editors.

genomics↗

bioRxiv2024

Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2+ human melanoma cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2+ melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E+ solid tumors.

bioengineering↗

bioRxiv2023

Base editing as a genetic treatment for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C*G-to-T*A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2, reverting the exon 7 mutation via an A*T-to-G*C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.

genetics↗

bioRxiv2022

IFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation

Activating mutations of KRAS play critical roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence indicates that distinct KRAS alleles associate with different prognoses, but the underlying mechanisms are not known. We established isogenic KRAS mutants (KRASG12D, KRASG12V, and KRASWT) using a KRASG12R patient-derived PDAC cell line by CRISPR/Cas9 knock-in. We used these isogenic cell lines, a collection of characterized human PDAC patient-derived cell lines, and murine PDAC models to study the role of these KRAS alleles in vitro and in vivo. We verified that the growth of KRASG12D cells is more aggressive compared to KRASG12V isogenic cells in vitro and in vivo using orthotopic mouse models. Signal transducer and activator of transcription (STAT) activation was the most significant difference between KRASG12D and KRASG12V isogenic PDACs. Furthermore, activation of interferon-alpha (IFNA)/IFNA receptor (IFNAR)1/STAT3 signaling in the cancer cells mediated the more aggressive phenotype of KRASG12D PDACs. Conversely, inhibition of IFNAR1 in patient-derived PDAC cells suppressed tumor growth. Finally, IFNAR1 blockade was also effective in murine PDAC models and induced a significant increase in survival when combined with immune checkpoint blockade therapy. We conclude that the IFNA pathway and IFNAR1/STAT3 axis contribute to a more aggressive tumor progression in human KRASG12D PDACs and that IFNAR1 inhibition is a potential therapeutic target for overcoming resistance to immunotherapy in PDAC. One Sentence SummaryIFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation.

cancer biology↗

bioRxiv2021

In vivo engineered B cells retain memory and secrete high titers of anti-HIV antibodies in mice

As a potential single-shot HIV therapy, transplanted engineered B cells allow robust secretion of broadly neutralizing antibodies (bNAbs). However, ex vivo engineering of autologous B cells is expensive and requires specialized facilities, while allogeneic B cell therapy necessitates MHC compatibility. Here, we develop in vivo B cell engineering, by injecting two adeno associated viral vectors, one coding for saCas9 and another coding for a bNAb. Following immunizations, we demonstrate memory retention and bNAb secretion at neutralizing titers. We observed minimal CRISPR/Cas9 off-target cleavage, using unbiased CHANGE-Seq analysis, while on-target cleavage at undesired tissues is reduced by expressing saCas9 from a B cell specific promoter. In vivo B cell engineering is thus a safe, potent and scalable method for expressing desired antibodies against HIV and beyond. One sentence summaryB cells can be engineered in vivo to robustly secrete anti-HIV bNAbs in a safe, durable and scalable manner.

immunology↗

bioRxiv2020

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

genetics↗

bioRxiv2020

Validation and Long-Term Follow Up of CD33 Off-Targets Predicted In Vitro and In Silico Using Error-Corrected Sequencing in Rhesus Macaques

ABSTRACTThe programmable nuclease technology CRISPR/Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR/Cas9 therapeutically. Here, we utilized a rhesus macaque model to ask whether CIRCLE-Seq (CS), an in vitro off-target prediction method, more accurately identifies off-targets compared to in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-Seq and ISP for guide RNAs designed against TET2 and CD33 genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both ISP and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a CD33 gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both ISP and CIRCLE-Seq, however there was poor correlation between the sites predicted by the two methods. When almost 500 sites identified by each method were searched for in hematopoietic cells following transplantation, 19 detectable mutations in off-target sites were detected via error-corrected sequencing. Of these 19 sites, 8 sites were predicted in the top 500 sites by both methods, 8 by CIRCLE-Seq only, and 3 by ISP only. Cells with off-target editing exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In conclusion, neither methodology predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods may be required for optimizing safety of clinical development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Zebrafish Dscaml1 is Essential for Retinal Patterning and Function of Oculomotor Subcircuits

Down Syndrome Cell Adhesion Molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the role of dscaml1 in the zebrafish oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Loss of zebrafish dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses showed that mutants have abnormal gaze stabilization, impaired fixation, disconjugation, and faster fatigue. Notably, the saccade and fatigue phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, for which no animal model exists. Two-photon calcium imaging showed that loss of dscaml1 leads to impairment in the saccadic premotor pathway but not the pretectum-vestibular premotor pathway, indicating a subcircuit requirement for dscaml1. Together, we show that dscaml1 has both broad and specific roles in oculomotor circuit function, providing a new animal model to investigate the development of premotor pathways and their associated human ocular disorders.

neuroscience↗

bioRxiv2018

In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1-5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8-12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

molecular biology↗