Locus Biosciences is a clinical-stage pharmaceutical company, founded in 2015 and based in Research Triangle Park, North Carolina which to develop phage therapies based on CRISPR–Cas3 gene editing technology, as opposed to the more commonly used CRISPR-Case9, delivered by engineered bacteriophages. The intended therapeutic targets are antibiotic-resistant bacterial infections.
Abstract Background LBP-EC01 is the first CRISPR-engineered bacteriophage product to successfully complete Phase 1b testing in a clinical program designed to address infections caused by E. coli initially targeting urinary tract infections (UTIs). Thirty-six subjects were enrolled in this randomized, double blind study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of LBP-EC01 in patients with lower urinary tract colonization caused by E. coli. Methods Results No drug-related Treatment Emergent Adverse Events (TEAEs) were observed. All nondrug related TEAEs were Grade 2 or below and there were no tolerability signals associated with LBP-EC01.A modified ITT (mITT) population was defined to assess PK in subjects treated with LBP-EC01 (n=17). Subjects were removed from the PK analysis who had missing or insufficient colonization at baseline (n=3), were exposed to antibiotics during screening or study conduct (n=3) or exhibited a non-drug related SAE (n=1). Of these subjects, 12 were found to be sensitive to LBP-EC01 and of these, 10 (83%) showed phage amplification. A PD analysis compared the mITT populations of LBP-EC01 vs. placebo (n=6) and showed that the LBP-EC01 arm had a decrease in E. coli that was greatest within 24 hours of initial treatment and remained below baseline across the entire treatment period. The placebo arm showed increased levels of E. coli and higher variability over the treatment period. An average difference of 2-3 log (100x to 1,000x) existed in urine E. coli concentration (CFU/mL) between the LBP-EC01 and placebo arms across the duration of the treatment period. Conclusion LBP-EC01 has proved to be safe and well-tolerated in this Phase 1b study of subjects colonized by E. coli. In addition, phage amplification was observed in patients with E. coli isolates sensitive to LBP-EC01, demonstrating a clear proof of mechanism. Finally, the apparent difference in PD effect between LBP-EC01 and placebo which was irrespective of MDR status, provides promise that LBP-EC01 may someday be an excellent option for patients suffering from UTIs caused by E. coli, especially in strains that are resistant to conventional antibiotics. Disclosures Dave ousterout, PhD, InceptorBio (Advisor or Review Panel member, Shareholder)Locus Biosciences (Employee, Shareholder)
Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile. Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms. IMPORTANCE Clostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broadspectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficile in vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be com- bined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.
The highly recombinogenic nature of human immunodeficiency virus type 1 (HIV-1) leads to recombination and emergence of quasispecies. It is important to reliably identify subpopulations to understand the complexity of a viral population for drug resistance surveillance and vaccine development. High-throughput sequencing (HTS) provides improved resolution over Sanger sequencing for the analysis of heterogeneous viral subpopulations. However, current methods of analysis of HTS reads are unable to fully address accurate population reconstruction. Hence, there is a dire need for a more sensitive, accurate, user-friendly, and cost-effective method to analyze viral quasispecies. For this purpose, we have improved the HIVE-hexahedron algorithm that we previously developed with in silico short sequences to analyze raw HTS short reads. The significance of this study is that our standalone algorithm enables a streamlined analysis of quasispecies, subtype, and recombination patterns from long HIV-1 genome regions without the need of additional sequence analysis tools. Distinct viral populations and recombination patterns identified by HIVE-hexahedron are further validated by comparison with sequences obtained by single genome sequencing (SGS).
