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CRISPR Nanomedicines for therapeutic and diagnostic applications

Dr. Deepak Chitkara is an associate professor at the Department of Pharmacy, Pilani Campus, BITS Pilani, and his lab is actively working on CRISPR/Cas gene editing tools for therapeutic and diagnostic applications. They are developing nanotechnology-based products to facilitate the efficient delivery of CRISPR for the treatment of debilitating diseases. The lab also focuses on developing CRISPR-based biosensors for early-stage diagnosis.

CRISPR/Cas9 is a molecular scissor that could provide precise and site-specific gene editing via double-strand break (DSB) mediated non-homologous end joining (NHEJ) or homology-directed repair (HDR) repair pathways (Figure 1a). The technology has shown immense potential as a therapeutic tool for treating uncurable genetic or non-genetic diseases. However, the use of the CRISPR/Cas tool is limited due to its high molecular weight, negative charge, and instability in the presence of nucleases or proteases, etc. (Figure 1b). Since 2017, Dr. Chitkara’s lab has been actively developing non-viral lipidic and polymeric nano-carriers to deliver CRISPR components, specifically the ribonucleoprotein (RNPs) complexes. We have developed lipo-polymeric carriers based on a polycarbonate backbone that stabilizes and effectively delivers these ribonucleoproteins to the cells and in animal models.

 Figure 1. a) Mechanism of gene editing by CRISPR/Cas9 and b) challenges in the delivery of CRISPR ribonucleoproteins
These systems could be modulated to target any gene of interest. For instance, it has been shown that the overexpression of vascular endothelium growth factor A (VEGF-A) leads to the progression of wet age-related macular degeneration (wet-AMD). Anti-VEGF antibodies are on the market but have several limitations, such as high cost, patient in-compliance, and resistance. Knocking out of the VEGF-A gene in the retinal cells in vitro and in vivo using CRISPR/Cas9 tool delivered using developed nanomedicines could have high therapeutic potential. The research outcomes showed that the CRISPR/Cas nanomedicine has a size of ~150 nm with a transfection efficiency of > 70%. The gene editing assay showed >40% VEGFA indel in mice and human cell lines. Moreover, nanomedicine has shown transfection of retinal cells in vivo in rats after intravitreal injection followed by VEGFA gene editing with an Indel frequency of ~10% (Figure 2).
 Figure 2. CRISPR Nanomedicine and its characteristic features 
Apart from editing the gene, epigenome editing using CRISPR/Cas tools is interestingly being used as a therapeutic approach to precisely modulate gene expression at the transcriptional level. Herein a catalytically inactive dCas9 fused with a repressor chromatin modifier provides a precise genetic modulation without causing DNA double-strand break. This strategy could be utilized for therapeutic purposes as well; for instance, diabetic nephropathy (DN), a frequent complication in diabetes mellitus, is considered one of the major causes of end-stage renal failure in patients. Despite current therapies, this aliment is inevitably reported to date. We are utilizing epigenome editing for genetic interrogation and modulation of Col 1 gene expression by CRISPR/dCas9-based epigenome targeting to treat diabetic nephropathy. However, for delivery purposes, we are engaging the developed nanomedicines.
For diagnostic applications, the CRISPR/Cas gene editing tool evolved swiftly, and, as of now, different Cas effector proteins have been discovered, each with distinct features and applications. Some of the most common chronic diseases include Chronic Kidney Disease (CKD), Cardiovascular diseases (CVDs), and Type 2 Diabetes Mellitus (T2DM). These diseases are life-threatening once they become severe. For instance, uncontrolled Diabetes Mellitus (DM) could eventually lead to several complications, including retinopathy, cardiomyopathy, and nephropathy. However, the management of these diseases is possible if the patient can be treated at an early stage. Biosensors are the tools that can diagnose any infection/disease by analyzing molecular biomarkers such as nucleic acids and proteins. The existing methods could not provide information about the onset or early stage information due to limitations associated with current approaches, such as sensitivity, cost, false positive/negative results, and unavailability of sophisticated instruments. Although efforts have been made to improve the existing technologies in terms of their sensitivity and accuracy, there is still a high requirement for the development of an alternative method for early-stage diagnosis of chronic disease.  

Figure 2. CRISPR dCas9 mediated epigenome editing to control gene expression

Recently, the Cas effector of class V of the CRISPR/Cas system, namely Cas12 and Cas13, are reported for their trans-cleavage activity that could be utilized for diagnostic applications. We are developing a CRISPR biosensor for early-stage diagnosis of diabetic nephropathy wherein the changes in the cell-free DNA (cfDNA) in the blood and urine could reflect the pathological condition and associated complications. CRISPR biosensors have been reported for their ability to detect nucleic acid at the femtomole level with high accuracy and precision. Therefore, they could be a potential method to detect nucleic acid biomarkers. Interrogation of the cell-free DNA isolated from the blood and urine samples could be used to diagnose a disease progression. Briefly, the Cas12 RNPs are designed to identify a specific gene in the cell-free DNA that could directly correlate with the disease progression (Figure 3).

Figure 3. CRISPR/Cas based biosensor for diagnostic applications

CRISPR technology has shown immense potential for therapeutic and diagnostic applications. Our lab’s focus is to innovate and translate advanced healthcare products on CRISPR technology for societal benefits.