CRISPBac-T (CRISPR Bacteria For Targeted Therapy) the in silico platform

CRISPR-based therapeutics face a significant challenge in restricting gene editing to diseased tissues. Since CRISPR enzymes target genes rather than specific cells, indiscriminate delivery can result in widespread apoptosis, toxicity, and potentially fatal off-target mutations, particularly when viral vectors or lipid nanoparticles are employed. Moreover, the intrinsic cytotoxicity of CRISPR enzymes introduces additional safety concerns. To address these issues, we propose a targeted delivery system utilizing genetically modified Escherichia coli Nissle 1917, a probiotic strain compatible with human physiology. These bacteria are engineered to thrive exclusively within pathological microenvironments characterized by hypoxia and low pH, and to recognize cancer-specific surface markers such as HER2 and CD44, thereby ensuring spatial precision. To circumvent cargo size limitations, the tol-pal gene will be incorporated to enhance vesicle-mediated export, while the lpp gene will facilitate the targeting of recombinant proteins into bacterial carriers. Additionally, the expression of inhibitor of apoptosis proteins (IAPs) aims to delay apoptosis and prevent inflammatory necrosis. This integrated design enables targeting at both the cellular and genetic levels. For early detection, the HSV1 thymidine kinase (TK) reporter gene permits positron emission tomography (PET)-based visualization of bacterial clusters, indicating the presence of a disease-permissive environment. Upon localization, CRISPR-Cas12a cleaves the TATA-binding protein (TBP) gene, resulting in transcriptional silencing and apoptosis specifically within malignant cells. This approach establishes a fully targeted, in vivo-applicable gene therapy platform. We hypothesize that genetically engineered bacteria will survive exclusively in diseased microenvironments characterized by low oxygen and acidic pH, while being eliminated in healthy tissues, thereby restricting gene editing to pathological sites. Furthermore, bacterial surface receptors targeting HER2 and CD44 will mediate specific adhesion to cancer cells. Guide RNA will direct the CRISPR- Cas12a complex to the TBP gene, facilitating precise cleavage with minimal off-target activity and enzyme leakage. Disruption of TBP is expected to induce gene silencing and subsequent apoptosis in cancer cells. Gene editing technologies such as CRISPR-Cas12a utilize RNA guides and nucleases to modify, cleave, or silence specific genes. Despite their potential, clinical application in diseases like cancer remains limited due to the risk of non-specific gene editing leading to cell death in healthy tissues. Current delivery methods, including adeno-associated viral (AAV) vectors and lipid nanoparticles, lack the spatial specificity required for safe therapeutic use. Effective gene therapy necessitates CRISPR delivery exclusively to cells harboring genetic abnormalities, avoiding healthy cells. To overcome these limitations, engineered bacterial delivery systems present a novel solution. The probiotic strain E. coli Nissle 1917 can be genetically programmed to activate CRISPR components solely within pathological conditions such as hypoxia and acidic pH. Additionally, bacterial surface receptors can be modified to recognize tumor-specific markers CD44 and HER2, enabling selective targeting of both cancer stem cells and differentiated cancer cells. Following CRISPR-mediated cleavage of the TBP gene, which plays a critical role in transcription, targeted cells undergo apoptosis. Expression of IAP proteins is incorporated to mitigate necrotic cell death and associated inflammation. This research aims to develop and validate a genetically engineered E. coli Nissle 1917 system capable of precise CRISPR-Cas12a delivery and activity restricted to tumor microenvironments, providing a safe and effective gene therapy platform for cancer treatment.