Blog Post

Genetic Modification of Blood Cells: The Here and Now of Nucleic Acid-Based Applications

**This Blog post has not been Peer Reviewed**

The genetic modification of blood cells has immense potential for clinical therapy since these cells serve as the foundation of both immune and hematopoietic systems [1]. However, the modification of these cells has been riddled with challenges making it historically difficult, explaining their limited use in treatment regimes. Recent advances in molecular biology have opened up new possibilities for the use of blood cells in therapy, mainly driven by utilizing innate mechanisms such as RNA interference (RNAi) and CRISPR/Cas system [2,3] that can engineer the cellular genome in a clinician- or user-defined manner to bring about transient or permanent physiological effects. The success of these gene-based therapies is contingent on the efficacy of nucleic acid delivery tools through all stages of development, that is, initial in vitro studies, preclinical (animal) studies and ultimately in clinical settings. This blog provides a brief overview of approaches for genetically modifying blood-resident cells from a therapeutic perspective, with an emphasis on nucleic acids deployed and issues pertaining to the transport of nucleic acids into cells. The importance of these therapeutic technologies is highlighted with a special focus on blood cancers.

Diseases of Blood Cells

A diverse population of cells resides in blood and circulating fluids including monocytes, lymphocytes, neutrophils, eosinophils, basophils, macrophages, erythrocytes, and platelets, all with the exception of erythrocytes and platelets being involved in host immunity [4]. Several malignancies stem from and afflict the immune and hematopoietic systems including autoreactive immune diseases such as rheumatoid arthritis and periodontis [5,6], and cancers of circulating fluids such as leukemias [7], myelomas [8] and lymphomas [9]. Hematological cancers arise as a consequence of genetic mutations in blood-resident cells and affect both children and adults [7,10]. There are over 137 known types of blood cancers which affect ~1 million individuals in the United States alone based on statistics from 2019 [11]. Blood cancers are especially devastating as most current treatment strategies do not prevent recurrence of disease and mortality rates remain dismally high based on cancer type [12]. Therefore, there is a pressing need for improved therapeutic agents capable of complete eradication of malignant cells to avoid recurrence from residual disease. Gene therapies are best suited towards this end as genetic manipulation of diseased cells to amend the disease-causing/propagating genetic alteration bears promising chance as a ‘cure’. The relevant cell populations are readily accessible in circulating fluids and hence delivery of active nucleic acids does not necessarily require custom engineering for site-specific targeting.

Main Approaches to Modifying Cells for Therapy

Several blood-resident cell types have been explored for therapeutic utility and different methods have been developed to engineer them for effective targeting and functional activity. Chimeric Antigen Receptor (CAR) expressing T-cells, also known as CAR T-cells, were the first clinically validated cell-based therapy as T-cells naturally possess the ability to target abnormal cells [2,7]. T-cells have been engineered to improve not only their targeting ability by promoting cytokine expression but also their longevity to enhance the effectiveness of the treatment. Native T-cells are transformed into CAR T-cells through the delivery of DNA or RNA to induce cell-surface CAR synthesis, typically with viral vectors [13] and synthetic DNA or mRNA molecules [14]. CAR T-cells capable of targeting antigens, such as CD19, in circulating cells have been shown to be highly effective in B-cell leukemias [15,16], and has instigated exploration in related hematological and solid cancers. Apart from T-cells, other blood-resident cells such as Natural Killer cells [17], platelets [18], hematopoietic stem cells [19], and hematopoietic stem progenitor cells [20] have been engineered and deployed for therapeutic use in cancers. In each of these cases, DNA or RNA molecules carried by appropriate delivery vehicles, to ensure entry of nucleic acids into cells, are employed to engineer the cells.

Aside from the use of viral vectors, tremendous efforts have been directed towards utilizing other nucleic acid-based technologies such as RNAi and CRISPR/Cas. RNAi uses small (19-27 bp) RNA oligonucleotides to downregulate protein synthesis by mRNA interference to facilitate cellular processes that aid in cancer eradication; this is an effective approach when the target is a protein essential for cell viability, so that it’s silencing can induce cell death [2,21]. The first siRNA drug candidate entered clinical trials in 2004 with the first siRNA drug being approved in 2018 [22]. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) can also be employed to execute gene knockout in cells to ‘permanently’ silence the expression of specific genes, in addition to gene editing to modify/restore function [20]. Although gene insertion via homologous recombination is possible, efficiency of proper insertion is low and therefore still too premature to implement as a therapeutic strategy [3]. CRISPR has been shown to be effective in engineering T-cells for treatment of HIV [3,23] and for reducing cytotoxicity in hematopoietic stem and progenitor cells [19,20]. Malignant cells in circulation have also been targeted with RNAi, leading to eradication of defective stem cells populating the circulation with immature cells [24,25].

Delivery Strategies for Effective Cell Modifications

Efficient delivery is paramount for ensuring the efficacy of nucleic acid therapeutics. This is especially important in blood diseases due to the challenge of introducing nucleic acids or their formulations into suspension-growing (in culture) and circulating (in vivo) cells. The inability to effectively deliver plasmid DNA, mRNA, siRNA, and CRISPR/Cas ribonucleoprotein (RNP) can drastically limit the advancement of potential treatments to clinical trials. Foreign nucleic acids are additionally vulnerable to degradation or systemic removal, thus restricting the use of naked nucleic acids in treatment regimens [26]. RNA-based therapeutic agents are particularly prone to nuclease degradation. Furthermore, the large size of nucleic acids and their anionic nature present major hurdles for infiltration into cells, and thereby require use of specially designed delivery vehicles or chemical modifications to evade nuclease degradation [21].

Polymers that possess multimeric binding sites to nucleic acids, lipids that display high affinity to lipidic cell membranes and metallic nanoparticles that exhibit unique features at the nano-scale [21-23] are among some of the relatively safe materials used as nucleic acid delivery tools. Each material varies in mechanism of nucleic acid assembly and encapsulation efficiency, due to differences in structural and chemical properties. However, they all attempt to provide attributes that enhance the ability of nucleic acids to move through the cell membrane, dissociate and release the cargo once in the cells, and provide a physical barrier against nuclease degradation. Many of these technologies are still in preclinical development, but lipid-based nanoparticles (liposomes and other variations) have emerged as the material of choice for clinical use since their FDA approval in 1995 [27]. However, lipid-based nanoparticles still face limitations such as poor encapsulation efficiency of hydrophilic drugs and leakage of cargo after assembly and drug deployment. Additionally, the transfection efficiency and ability to encapsulate and deliver different types of cargo varies based on the nature of nucleic acid and features of target cell type. Thus, a superior platform is required to refine and advance nucleic acid therapeutics.

To facilitate RNAi and CRISPR/Cas9 editing in blood cells, carriers tailored for encapsulation and delivery of specific nucleic acids are crucial. Optimizing delivery of nucleic acids is the first step towards implementing personalized medicine and introducing new treatments for a wide range of blood diseases. RJH Biosciences is dedicated to finding solutions for nucleic acid delivery issues in different cell types for an array of different applications. RJH Biosciences has designed and developed a unique category of lipophilic material called cationic lipopolymers which possess favorable characteristics of both polymer and lipid-based nucleic acid delivery vehicles. Cationic lipopolymers are highly effective in delivering siRNA to suspension cells, which by nature display minimal endocytosis, and provide robust protection against degradation. This allows native nucleic acids to be used for therapy, rather than chemically modified versions which maintain integrity of nucleic acids in physiological conditions but may adversely affect potency. RJH formulations provide superior delivery of CRISPR pDNA and Cas9-sgRNA complexes as seen in initial characterization studies using Jurkat T-cells.

As cell-based therapies become more prevalent on the market, the choice of expression system (e.g., plasmid DNA, mRNA, transposon, etc.) and the delivery method for the deployed nucleic acids become key in ensuring proper genetic modification of cells intended for therapy. While direct (in situ) modification of such cells is limited by physiological factors, one can attain increased flexibility for cell engineering under culture conditions; the cellular media can be adopted for optimal transfection, and the modified cells can be selected/expanded to propagate pure populations for host administration. Moving beyond lipidic systems and exploring other delivery vehicles such as cationic lipopolymers can lead to paradigm-shifting therapeutic innovations in the realm of blood cell modifications.

For more information on our nucleic acid delivery technology, feel free to reach out to us at rjhbiosciences@gmail.com.

References:

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