Biology subjects

Rabuka, D.

Publications and source records attributed to Rabuka, D..

7 recordsLinked to original sources

bioRxiv2024

Viral delivery of compact CRISPR-Cas12f for in vivo gene editing applications

Treating human genetic conditions in vivo requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with ex vivo therapies and with in vivo delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body. However, the large size of most CRISPR-Cas systems limits packaging into the viral genome and reduce drug development flexibility and manufacturing efficiency. Here, we demonstrate efficient CRISPR gene editing using a miniature CRISPR-Cas12f system with expanded genome targeting packaged into AAV particles. We identified efficient guides for four therapeutic gene targets and encoded the guides and the Cas12f nuclease into a single AAV. We then demonstrate editing in multiple cell lines, patient fibroblasts, and primary hepatocytes. We then screened the cells for off-target editing, demonstrating the safety of the therapeutics. These results represent an important step in applying in vivo CRISPR editing to diverse genetic sequences and organs in the body.

molecular biology↗

bioRxiv2023

Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications

The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We then engineer the nuclease (termed AiEvo2) for increased specificity, PAM recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntingtons patient derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 CAR-T therapy from primary human T cells using multiplexed simultaneous editing and CAR insertion. To further ensure precise editing, we engineered an anti-CRISPR protein (ErAcr) to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel ErAcr protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.

biochemistry↗

bioRxiv2023

Anti-CRISPR protein mediated degradation of Cas9 in human cells

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9. Here we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. Optimized expression of AcrIIA1 in human cells provided robust inhibition of SpyCas9 editing but had no impact on Type V CRIPSR-Cas12a, consistent with its binding site on the HNH domain. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

biochemistry↗

bioRxiv2023

Casμ, a novel class of miniature type-V Cas12f nucleases with diverse PAM

Small CRISPR-Cas effectors are key to developing gene editing therapies due to the packaging constraints in viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type-V Cas12f effectors present a solution given their small size. Here we describe Cas, a novel class of miniature (<490AA) type-V Cas12f nucleases that cleave double stranded DNA in human cells. We determined their optimal trans-activating RNA (tracrRNA) empirically through rational modifications which resulted in an optimal single guide RNA (sgRNA). We show that the Cas nucleases have broader PAM preferences than previously known Cas12f effectors. The unique characteristics of these novel nucleases add to the diversity of the miniature CRISPR-Cas toolbox while the expanded PAM allows for the editing of genomic locations that could not be accessed with existing Cas12f nucleases.

biochemistry↗

bioRxiv2021

Tandem-Cleavage Linkers Improve the In Vivo Stability and Tolerability of Antibody-Drug Conjugates

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, leading to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically-encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, liberating the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system, and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results--where rat studies showed dramatically improved tolerability in the hematopoietic compartment--highlight the role that linker stability plays in efficacy and tolerability, and offer a means of improving an ADCs therapeutic index for improved patient outcomes.

pharmacology and toxicology↗

bioRxiv2020

A novel HER2-targeted antibody-drug conjugate offers the possibility of clinical dosing at trastuzumab-equivalent exposure levels

Trastuzumab and the related antibody-drug conjugate (ADC), ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2+ breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload. Interestingly, in the clinic, T-DM1 cannot always replace the use of trastuzumab plus chemotherapy administered together as single agents. We hypothesize that this failure may be due in part to the limited systemic exposure achieved by T-DM1 relative to trastuzumab because of toxicity-related dosing constraints on the ADC. We have developed a trastuzumab-based ADC site-specifically conjugated to maytansine through a noncleavable linker. This construct, termed CAT-01-106, has a drug-to-antibody ratio (DAR) of 1.8, approximately half the average DAR of T-DM1, which comprises a mixture of antibodies variously conjugated with DARs ranging from 0-8. The high DAR species present in T-DM1 contribute to its toxicity and limit its clinical dose. CAT-01-106 showed superior in vivo efficacy compared to T-DM1 at equal payload dosing and was equally or better tolerated compared to T-DM1 at equal payload dosing up to 120 mg/kg in Sprague-Dawley rats and 60 mg/kg in cynomolgus monkeys. CAT-01-106 also showed improved pharmacokinetics in rats relative to T-DM1, with 40% higher ADC exposure levels. Together, the data suggest that CAT-01-106 may be sufficiently tolerable to enable clinical dosing at trastuzumab-equivalent exposure levels, combining the functions of both the antibody and the payload in one drug and potentially improving patient outcomes.

pharmacology and toxicology↗

bioRxiv2019

Systematic identification of regulators of antibody-drug conjugate toxicity using CRISPR-Cas9 screens

Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years. However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisingly appear to bypass the requirement of lysosomal delivery. Lastly, we show that inhibition of sialic acid biosynthesis sensitizes cells to ADC treatment by increasing the rate of ADC internalization. This sensitization was observed using several ADCs targeting different antigens in diverse cancer cell types, including the FDA-approved ADC trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal novel regulators of endolysosomal trafficking, provide important insights to guide future ADC design, and identify candidate combination therapy targets as well as potential mechanisms of ADC resistance.

cancer biology↗