Delivery systems of CRISPR/Cas9-based cancer gene therapy

Clustered Regularly Interspaced Short Palindromic Repeats Cas9 endonuclease (CRISPR/Cas9) is a powerful gene editing tool with immense potential in targeted DNA modifications[1, 2]. It uses guided RNA (gRNA) that directs the Cas9 enzyme to introduce specific edits in the DNA [3]. This is preceded by the cell’s natural mechanism of introduction of desired genetic traits via insertions or deletions. Because of this unique application, this technology has been used as a gene therapy in managing many diseases including cancer.

However, to make DNA alterations, CRISPR/Cas9 components have to reach target DNA sequences within the cell. Various delivery methods have been employed which include viral vectors, lipid nanoparticles, and physical techniques [4]. In the context of cancer, CRISPR/Cas9 offers a strategy to induce specific alterations in cancer-related genes, thereby correcting genetic defects, and inactive oncogenes, or restoring the function of tumor suppressor genes.

This article aims to explore various delivery system methodologies utilized in CRISPR/Cas9-based cancer gene therapy.

Viral delivery systems in CRISPR/Cas9-based cancer gene therapy

It is important to deliver the CRISPR/Cas9 components into the target cell, for which various delivery systems are in place. Delivery systems like adeno-associated viruses (AAVs), lentiviruses, and adenoviruses are the most commonly used viral-based delivery systems. Lentiviruses were the first to be adopted for genome-editing applications [5]. Owing to their safety profile and ability to transduce both diving and non-diving cells, make them accurate delivery vehicles.

Likewise, Adenovirus possess a high transduction and packaging capacity, that makes them suitable for delivering CRISPR/Cas9 constructs [7]. Moreover, they do not integrate into the host genome, reducing the risk of insertional mutagenesis. Recent literature evidences the role of viral-mediated CRISPR/Cas9 delivery in treating liver cancer, glioblastoma, and other malignancies [8-10]. Although viral delivery vehicles have proven efficient, they possess a few limitations.

Moreover, their modest packaging capacity limits the amount of genetic material that can be delivered, which can be difficult when larger CRISPR/Cas9 constructions are required.

Advantages of viral vectors in gene therapy

Viral vectors are effective due to their natural ability to infect and integrate their genetic content into host cells. This distinctive feature makes them a good choice for efficient delivery of the CRISPR/Cas9 components. They can be programmed to target specific types of cancer cells by altering their surface proteins or adding targeting ligands [11]. They also facilitate the packaging of large constructs with multiple gRNAs or additional regulatory elements [12]. They can be engineered to reduce immunogenicity which decreases the likelihood of adverse immune responses against the vector or the delivered therapeutic agents.

Although viral delivery systems of CRISPR/Cas9 constructs have shown immense potential in cancer gene therapy, they come with some safety concerns. Immunogenicity is one of the prime safety concerns associated with viral delivery systems in CRISPR/Cas9-based cancer gene therapy. Unless a viral vehicle is rendered non- or less immunogenic, chances of eliciting immune responses are higher. Insertional mutagenesis is another safety issue that poses a risk of activating oncogenes or causing unintended genetic alterations [13]. Efforts are needed to minimize these associated risks and make the viral delivery system safe and secure.

Non-viral delivery systems

Non-viral delivery systems are also used in delivering CRISPR/Cas9 components. These methods come in various options.

Each method is utilized based on the delivery cargo and the target cell type. For instance, physical methods like electroporation or hydrodynamic delivery have been used while targeting multiple sites simultaneously [17]. Likewise, targeted delivery strategies like Ligand-receptor, antibody-conjugates, or aptamer-based targeting bind are designed to target specific cancer cell markers, like FR – α and β which are overexpressed in many epithelial cell cancers [18].

Similarly, electroporation has been used to deliver CRISPR/Cas9 components in targeting transferrin monomeric glycoprotein genes. This gene is highly expressed in certain cancers like kidney and breast [19]

Non-viral delivery systems offer several distinct benefits. They are less immunogenic or may not generate any sort of immune response when compared to viral-based vectors. Additionally, they do not integrate into the host genome and, hence possess minimal risk for insertional mutagenesis [20]. Moreover, their production is less costly and does not involve much of a biological system, which makes them scalable and sustainable.

Challenges such as efficiency and targeting

Despite owning some benefits over viral-based vectors, non-viral-based vectors have certain limitations and efficiency challenges. Non-viral delivery systems struggle to penetrate through cell membranes, making CRISPR/Cas9 component delivery challenging [21]. This results in suboptimal gene editing outcomes. Non-viral systems often lack the natural tropism of viral vectors, which means they may not specifically target the intended cells or tissues [2 2 ]. This leads to off-target effects and reduced therapeutic efficacy. Additionally, non-viral delivery systems face stability and release issues.

Irrespective of the delivery system used, it is important that CRISPR/Cas9 accurately edits only the intended genetic sequences, thereby ensuring minimal off-target effects that could potentially disrupt normal gene function. Specificity and efficiency issues do persist in delivering CRISPR/Cas9 components to target cancer cells. Non-viral delivery technologies, such as lipid nanoparticles and polymer-based carriers, frequently exhibit low transfection efficacy and limited capacity to precisely target specific cells or regions. While techniques like ligand-receptor targeting and antibody conjugation are being developed to enhance specificity, settling high targeting precision with effective delivery remains a challenging problem. The use of CRISPR/Cas9 systems, regardless of the delivery route can result in unwanted genomic changes or immunological responses. Non-viral methods while less immunogenic than viral ones, can however cause local or systemic toxicity. Ensuring that CRISPR/Cas9 components are administered safely and with minimum side effects is critical for successful therapy.

Ethical considerations should be kept in place while developing CRISPR/Cas9-based treatments. The possibility of unintended genetic changes raises ethical concerns, particularly in human germline cells. These modifications may create a pool of undesired genetic traits that may have adverse consequences for future generations.

CRISPR/Cas9-based cancer gene therapy has opened new avenues in treating different cancers. Effective delivery systems are essential to achieve this goal. While viral vectors have confirmed significant efficacy, non-viral systems offer promising alternatives such as reduced immunogenicity and prolonged scalability. Mitigation of challenges like immunogenicity, toxicity issues, and insertional mutagenesis associated with delivery systems needs careful consideration to ensure that CRISPR/Cas9 components are delivered effectively, and safely, with minimal risk of adverse effects.

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