Prospects of Non-Coding Elements in Genomic DNA Based Gene Therapy

Abstract

Gene therapy has made significant development since the commencement of the first clinical trials a few decades ago and has remained a dynamic area of research regardless of obstacles such as immune response and insertional mutagenesis. Progression in various technologies like next-generation sequencing (NGS) and nanotechnology has established the importance of non-coding segments of a genome, thereby taking gene therapy to the next level. In this review, we have summarized the importance of non-coding elements, highlighting the advantages of using full-length genomic DNA loci (gDNA) compared to complementary DNA (cDNA) or minigene, currently used in gene therapy. The focus of this review is to provide an overview of the advances and the future of potential use of gDNA loci in gene therapy, expanding the therapeutic repertoire in molecular medicine.

1. INTRODUCTION

Gene therapy is an experimental approach where a therapeutic gene is used to treat or prevent various disorders, including monogenic and polygenic diseases and cancers. The basic concept of gene therapy involves the identification of a mutant gene, which is responsible for a disease and administering a therapeutic gene or transgene into the patient’s target cell to reinstate its normal function or to make a beneficial protein and cure the disease [1]. The transgene inside the target cell gets integrated into a chromosome or remains an episomal DNA and corrects the defective or mutated gene. Gene Therapy was born in 1962 when Szybalski introduced a foreign DNA in a mammalian cell and corrected a genetic defect [2]. Later in 1989, the first clinical gene delivery study by National Institutes of Health (NIH) proved genetically engineered tumor-infiltrating lymphocytes tagged using a retroviral vector can be reinfused safely to assess their tumor-homing ability [3]. In recent years, transformative advances in the field of gene therapy produced highly publicized clinical trials, the majority of which address cancer, inherited monogenic diseases, cardiovascular diseases, and infectious diseases (Fig. 1) [4, 5].

Fig. (1).

Graphic diagram showing various gene therapy clinical trials conducted worldwide to 2018. (Latest updates from [4, 5]).

Gene therapy is primarily divided into germ line and somatic cell gene therapy based on the tissues or cells that are targeted for gene delivery. Germ-line gene therapy is done by manipulating egg or sperm cells to make heritable changes that pass down permanent modifications to subsequent generations, which has been successfully validated in many species including mouse, rat, rabbit, sheep, cattle, goat, and pig, but not in humans due to ethical issues and societal consensus [6, 7]. In somatic gene therapy, the therapeutic entity is delivered into the target cells, making this approach more efficient and safe with minimized immune response. There have been several trials on hepatocytes in the liver, retina photoreceptors in the eye, stem cells in the bone marrow, and T lymphocytes that have used this approach [8], In this review, our focus is on the somatic cell gene therapy that is classified into two categories: ex vivo and in vivo. In the ex vivo delivery, cells are harvested from the patient, tailored with a therapeutic gene outside the body, and re-introduced into the body by systemic injection or local implantation. In vivo delivery involves introducing therapeutic genes via viral or non-viral vectors directly to target cells or tissue inside the body. This approach is broadly categorized into two types: viral vectors and non-viral vectors. Gene delivery using a viral vector is based on the viruses’ natural ability to transduce the genetic material into host cells, which makes them a highly efficient delivery system in gene therapy [9]. Some commonly used viral vectors include adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus.

Non-viral vectors constitute of physical methods, such as mechanical massage, needles, gene guns, electroporation, hydroporation, photoporation, ultrasound, magnetofection, and chemically fabricated biocompatible materials, such as nanoparticles and polymers [10]. They are considered safe and less prone to induce mutation and immune responses. However, the external and internal barriers and the low transfection efficiency are some key points that still need to be addressed in order to make them more beneficial than existing viral vectors [11, 12].

In addition to the criteria noted above, the most important requirement for successful gene therapy is persistent tissue-specific expression of the transgene. Most of the gene therapy trials use complementary DNA (cDNA) or minigenes, such as transgenes that are devoid of non-coding regions like 5’ flanking sequence (FS), introns, or the 3’ FS, which are limited by the relevant level of gene expression physiologically. Indeed, many researches being conducted focus on the delivery of complete locus of the genomic DNA (gDNA) of interest, which includes native regulatory elements (e.g., introns and 3’ and 5’ FS), promoter elements, and enhancer elements, showing substantial increase in stable transgene expression and avoiding random integration into the host genome [13, 14]. However, the major challenge in using gene full-length for gene therapy is to identify a vector that can carry the large gDNA (>10kb) but does not disturb or compromise the natural function of the gDNA and does not elicit a host immune response. This review aims to provide a cursory background on gene therapy strategy using gDNA loci with non-coding elements and their importance in various diseases that include cancer, genetic disorders, and neurological diseases. We have also briefly discussed the recent advances in NGS and nanotechnology, and its significance in relation to non-coding elements in the genome and the advantages of using full-length gDNA loci in gene therapy.

2. IMPORTANCE OF NON-CODING DNA IN GENE THERAPY

In this technology driven era, computational data analysis involving various high-throughput sequencing technologies has shifted the paradigm of the genome, from presence of only known discrete hereditable coding entities, to the presence of mysterious elements of the genome that might carry out some important biological function. The Human Genome Project completed in 2003 helped to launch several major projects like International HapMap Project [15], the 1000 Genomes Project [16], the Encyclopedia of DNA Elements (ENCODE) project [17, 18], and the ROADMAP project [19], generating unprecedented evidence on the human genome. These advances in the domain of genomic sequencing demonstrate that only about 1.5% of the total human DNA carries the information for all the genes through the production of mRNA; the other 98.5% of the total DNA is recognized as non-coding DNA [20]. Detailed reports gathered from the ENCODE project mapped regions that do not match protein coding regions but are enriched with regulatory functions like transcription, transcription factor association, chromatin structure, and histone modification [18]. Furthermore, in depth investigations revealed that the non-coding part of the genome, previously dismissed as “junk” DNA, is not junk after all. They are associated with functional elements consisting of promoters, enhancers, silencers, insulators, or locus control regions, from prokaryotes to eukaryotes. These regions are found to be involved in providing binding sites for the gene to carry out transcription, activating, repressing, and controlling transcription acting as enhancer, blocker, and barrier insulators [21].

Many repeated DNA segments like centromeres, transposable elements, satellite DNA, telomeres, and introns were present in the non-coding regions revealing vital structural and functional role at the chromosomal level. Besides, it was also found that certain kinds of functional non-coding RNA molecules involved in gene expression were found to have many key regulatory functions. These non-coding RNA (ncRNA) are broadly classified into small ncRNA associated with 5′ or 3′ regions of protein-coding genes including miRNAs, small-interfering RNAs (siRNAs) and PIWI-interacting RNAs), and long ncRNA similar to mRNA including linear RNA (lincRNA) and circular RNA (circRNA). The importance of these non-coding segments in cutting and splicing in transposon reassembly, genome rearrangements, and the production of small RNAs that serve as a source for new exons is ambiguous, thereby this is a flourishing area of research. Moreover, evidence prove that alterations in these non-coding regions is linked to many diseases. In this review, we provide a concise overview of the importance of various non-coding segments of the DNA with particular focus on Cis- and Trans-regulatory elements, Pseudogenes, Satellite DNA, Telomeres, and Introns in gene regulation, focusing on their functional role on regulating of gene expression and their potentials to be an essential tool in gene therapy.

3. CIS- AND TRANS-REGULATORY ELEMENTS

Cis-regulatory elements (CREs) are non-coding DNA sequences with binding sites for transcription factors located in or near a gene and are essential for proper gene expression. The CREs are classified based on binding sites in transcription as the promoter, enhancer, silencer, and insulator. They are involved in activating transcription, repressing transcription, and controlling transcription acting as enhancer-blocking insulator and barrier insulator. Trans-acting elements are non-coding DNA sequences that control the transcription of a distant gene [22]. They modify gene expression involved in regulation of transcription by binding to the CREs. They are involved in binding promoters necessary for initiation, acting as positive regulators and act as subunits of RNA polymerase, and stabilize the initiation complex. Studies have shown that cis and trans regulatory elements play a major role in gene transcription [23, 24], especially in the brain where there is high cis regulation [25]. A recent study evaluated and characterized the cis- and trans effects and their involvement in the regulating human -insulin receptor gene [24] and evolution of gene expression [26].

Mutations in such important regulatory elements disrupt the normal function of the cell and cause many disorders which do not have any effective treatment. For example: in various retinal diseases, photoreceptor (PR) degeneration is triggered by mutations in developmental regulators such as the transcription factors cone-rod homeobox (Crx) and neural retina leucine zipper (Nrl), causing defective PR differentiation during development [27], and proper forebrain development [28, 29]. In addition, many studies identify and characterize several cis-regulatory elements in retinal gene targeting and emphasize their importance in transgene expression [30]. Researchers have also found the cellular tropism of the CREs (enhancers/promoters) to be important for accurate delivery of the transgene to the target for an efficient expression of the rescue transgene [31, 32]. A study showed that using photoreceptor specific CREs can improve [33] and drive cell type-specific transgene expression [34, 35]. These studies emphasize the important role of CREs in driving cell-type-specific expression in gene therapy experiments, which highlights the need to explore the future of these elements in gene therapy.

4. PSEUDOGENES

Pseudogenes are DNA sequences that are like the parental gene but do not show normal protein coding functionality. Yet, small changes in these segments are associated with many diseases like cancer. Pseudogene transcripts regulate small RNA, lncRNA, and are involved in sequestrating trans-acting RNA decay proteins acting as a sponge for endogenous miRNAs and in addition play important role in recruiting epigenetic modifying complexes [36]. It has been reported that pseudogene PTEN can regulate different cellular events, including epigenetic, transcriptional, post-transcriptional mechanisms and post-translational modification [37]. Evidence has indicated that pseudogene PTEN play a key role in tumor suppressor activity in a many cancers, including breast cancer [38], esophageal squamous cell carcinoma [39], oral squamous cell carcinoma [40], head and neck squamous cell carcinoma [41] and melanoma [42] and renal cell carcinoma [43]. Studies using microarray indicate that pseudogenes (HSP90AA1 and HSP90AA2) and other miRNAs play an important role in the transcriptional level in cancer regulation [44, 45]. Other studies have indicated that pseudogenes have played a role in the regulation of tumor gene expression, including Foxo3P in breast cancer [46, 47], BRAF in thyroid cancer, [48, 49] DUXAP10 in colorectal cancer [50], and FTH1 in prostate cancer [51] Reports also show pseudogene-transcribed lncRNAs are emerging as both imperative regulators [52] of gene expression and as a potential unique target for pharmacological intervention in cancer gene therapy [53–55], increasing the use of pseudogenes in the development of therapeutics.

5. SATELLITE DNA

Satellite DNA are highly repetitive tandem sequences in the non-coding genome with several distinct biological functions including transcriptional activity during embryogenesis. They are mainly categorized based on individual repeats. Satellites are tandemly repeated sequences (>100 bp), are repeats sequences (>10 and <100 bp), short tandem repeats (STRs) are sequences that are 3 to 5 bp of size, and microsatellites are short repeat units (usually <10 bp) [56]. There are numerous studies depicting the vital role of satellite DNA repeats that form the centromere locus and heterochromatin of the pericentromeric area in chromosome organization, pairing, and segregation [57–59]. Besides, they are essential for assembly of the kinetochore, controlling telomere elongation, capping, and replication. They also modulate gene expression by epigenetic regulation and transcriptional response during stress. Many reports validate the use of murine satellite DNA-based artificial chromosomes (SATAC) as a functional mammalian artificial chromosome which can be used in gene therapy for stable long term gene expression, preserving their structural and functional integrity in vivo [60]. SATACs are the only mammalian artificial chromosomes that can be produced in large scale [61] Furthermore, SATAC has been used to generate transgenic animals and is used for germ line transmission [62]. Currently, satellite DNA-based artificial chromosomes are used as vectors in several gene therapy studies for delivering large gDNA with regulatory segments [60, 63, 64], implicating the significance of these non-coding segments in gene therapy.

6. TELOMERES

Telomeres are specialized nucleoprotein structures located at the end of each chromosome arm in eukaryotes for safe-guarding genetic stability and integrity. Various mutations or deletions of telomerase components have shown to cause life threatening inherited genetic disorders, resulting in the culmination of chromosomal abnormalities. In the recent past, numerous telomere gene therapy studies have provided valuable insights into the role of telomeres in premature aging disorders like Hutchinson-Gilford Progeria Syndrome (HGPS), Down’s syndrome, Dyskeratosis Congenita, Cockayne Syndrome, Nijmegen Breakage Syndrome, and Ataxia telangiectasia [65–71]. Several studies reveal the association of shortened telomeres in cardiovascular diseases, including atherosclerosis, hypertension, vascular dementia, and coronary heart disease, indicating its prospective role in gene therapy [72]. Evidence also shows downregulation of telomere-related genes have played an important role in cancer, emphasizing its possibility as an attractive tool in cancer gene therapy [73].

7. INTRONS

Introns, first discovered in 1977, are non-coding genomic sequences of an RNA transcript, or the DNA encoding it, that are spliced or removed before the RNA molecule is translated into a protein [74]. All higher organisms have introns, and more complex organisms have higher amounts of introns, indicating that they are involved in many diverse complicated functions. The most abundant class is spliceosomal introns, located in the nuclear genomes of eukaryotes consisting of RNAs and hundreds of proteins. These non-coding sequences present in mRNA, generally outsizes exons, are involved in differential joining of exons during splicing, consequently synthesizing proteins with new properties. They are also processed into non-coding RNAs that are involved in a wide variety of gene regulatory processes such as polyadenylation, transcription, translational efficiency, and in mRNA stability. Researchers have found that genes contain stimulating intron encode proteins that are required in large amounts for most cells, such as ubiquitin [75, 76], actin [77, 78], tubulin [79, 80], ribosomal proteins [81, 82] and elongation factors [83, 84].

The presence of endogenous non-coding sequences like introns and cis-acting elements in the genomic loci are essential for stable transgene expression, improving the transcriptional efficiency, and improving or reducing the translational yield as required [85–87]. Introns are seen in higher amounts in eukaryotes, thereby playing critical regulatory functions in the gene expression. Numerous experiments carried out in strains like Caenorhabditis elegans and Arabidopsis demonstrate that introns are more essential than the promoter for gene expression [85]. For instance, there is a more severe decline in the gene function when introns are removed from the genomic construct than when the promoter in C. elegans unc-54 mutant is deleted [88]. Another similar experiment was conducted in Arabidopsis, where deleting a 303 nt region did not instigate any appreciable decrease in the expression of TRP1:GUS reporter gene fusions as long as it had a stimulating intron within the coding sequence [89]. In support of the above experiments, insertion of an expression-stimulating intron into a gene that is usually active only in certain cell types showed to dominate the regulation offered by the promoter [78, 90], stressing the imperative role of introns in the genome.

Many experiments validate the influence of introns in gene expression providing details of the synergistic interactions of introns [91]. Previous studies in our laboratory compared delivery of a rhodopsin cDNA construct with an intron-containing construct in a rhodopsin knockout mouse model, establishing the beneficial role of introns in gene regulation with more lasting and higher physiological expression than its cDNA counterpart [92]. The constitutive durable gene expression demonstrated by the presence of introns in the gDNA loci makes it an ideal candidate for gene therapy. Altogether the contribution of these intervening sequences in gene regulation has shown research on introns to be effectual and remarkable in gene therapy.

8. ROLE OF NON-CODING ELEMENTS IN DISEASES

Progress in large consortia like ENCODE, the Epigenomics Roadmap coupled along with NGS, have aided in identifying and analyzing the etiology of diseases that are associated with non-coding elements. Following this study, several lines of evidence show that they play an important function in the regulation of gene activity by governing the on and off machinery of certain genes. Alteration in these sequences by mutation will either cause an incorrect protein to be expressed or complete absence of a protein, which is indispensable for normal function. Mutations in these sequences can trigger deleterious effects, disrupting normal development and causing genetic diseases. Genetic information flows from DNA to proteins, where RNA plays the intermediary role in the cascade [93]. Many reports on non-coding RNA and its function at the cellular level have added a new dimension, shedding light on the rich repertoire of its function role in many diseases like cancer [94], neurological disorders [95], congenital diseases, and cardiomyopathies [96].

Non-coding mutation in the promoter of the TERT gene, encoding the catalytic subunit of telomerase, is found to be associated with a wide variety of diseases including melanoma, low-grade glioma, medullo blastoma, colon cancer, parkinson disease, autism, and cleft lip with or without cleft palate [97–100]. Certain non-coding mutation reported in the DAAM1 gene makes tumor cells more aggressive by invading the surrounding tissues [101]. There is also evidence demonstrating that Type 2 diabetes (T2D)-associated variants are not seen in the coding region advocating the influence of non-coding elements on the genome [102]. Developmental disorders such as isolated Pierre Robin sequence are linked with mutation in the enhancer elements that regulate the activity of the SOX9 gene [103]. Non-coding lesions upstream of SOX9 are said to be connected with Acampomelic Campomelic Dysplasia (ACD), Brachydactyly Anonychia, and disorders of sex development (DSDs) during the development of the embryo, suggesting a complex regulatory domain that controls tissue-specific expression of SOX9 [104]. Preaxial polydactyly 2 (PPD2) was found to be caused due to a balanced translocation in an enhancer element [105, 106], the breakpoint of which is within intron 5 of the LM-BR1 gene. Pancreatic and cerebellar agenesis (PACA), a neonatal lethal disorder found in families with recessive mutations in the PTF1A (pancreas-specific transcription factor 1a) gene, is another example of mutation in the enhancer elements [107]. Non-coding mutations are observed in PAX6 gene causing aniridia [108], and another deletion mutation in DYNC1I1 gene due to deletion of exonic enhancer DLX5/6 is associated with split hand and foot malformation [109, 110]. Several other point mutations and duplications in the regulatory Sonic hedgehog (SHH) locus result in congenital limb malformations causing additional digits (polydactyly), fusion of digits (syndactyly) [106, 111], shortening of the digits (brachydactyly), and craniosynostosis [111, 112]. Non-coding mutations in RPGRIP1 cause inherited retinal degenerations (IRDs) [113], indicating the importance of non-coding elements in human diseases. Nonetheless, much is still unknown about how to identify and explain the role of functional regions of non-coding DNA, making it a very difficult task to link genetic changes in these regions to their effects in diseases. Mutations in the non-coding elements substantiate the importance of these regions, thereby urging more detailed study to aid medical interpretation and develop therapeutic measures which can be accomplished with gene therapy.

9. GENOMIC DNA VS COMPLEMENTARY DNA

Effective gene therapy not only depends on choice of delivery vehicle, but it is also significantly subject to the transgene selected. Currently, most of the gene delivery systems utilize a synthetic cDNA that can fit into viral vectors such as AAV, which has presented some encouraging results. These cDNAs or minigenes are short sequences devoid of the native gene’s regulatory sequences, such as the endogenous promoter, introns, enhancers, and poly(A). However, when these cDNA transgenic constructs were used to generate transgenic animals, they did not have required native gene function as expected [87, 114]. Importance of using genomic constructs with introns and other non-coding regions, like 5 -FS,3 -FS in transgenic animals is explained in detail in many studies showing their ability to enhance nuclear mRNA stability and transport in the assembly of spliceosome complex, giving the transgene similar chromatin conformation [87, 115–117]. In support of these experiments, mini introns in cDNA elevated the production of recombinant protein [118, 119] and enhanced transcriptional efficiency in transgenic mice [118, 120, 121]. Several other experiments in transgenic animals have also demonstrated that the use of non-coding segments eliminated various shortcomings seen in cDNA, such as transgene silencing, lack of crucial regulator elements, position effects, and down regulation by adjacent chromatin [14, 122]. Numerous reports have revealed that cDNA transgene and plasmid often cause transgene silencing which affects protein function due to overexpression [123, 124], which is overcome by using large gDNA transgenes [14]. A comparative study of the pattern of tyrosine hydrolase (TH) gene expression in rat brain and glial cells revealed increased and extended gene expression using genomic forms of TH gene that surpassed cDNA forms of TH gene [115]. Despite many positive results using cDNA for gene therapy, they failed to produce physiologically relevant level of gene expression in cells. For example, a study using cDNA coding for frataxin (FXN), for the treatment of a neurodegenerative disease known as Friedreich’s ataxia FRDA, produced toxic effects in some experimental models due to frataxin overexpression, indicating that it was not an optimal approach of gene therapy. In contrast, using 135kb entire (FXN) genomic locus for the treatment showed enhanced expression of the different FXN isoforms both in vitro and in vivo overcoming the drawbacks of using the cDNA counterpart [125]. Another study was conducted, where a 124kb full length genomic locus of human low-density lipoprotein receptor (LDLR) was delivered with improved and regulated gene expression in vivo, and demonstrated an effective gene replacement strategy in gene therapy for familial hypercholesterolemia (FH) [114, 126]. Several other lines of evidence show that using genomic locus as transgenes protects the cells from deleterious effects caused due to overexpression and provides physiological relevant and long term persistent regulated transgene expression [127–129]. We conducted several experiments that emphasize the importance of gDNA that not only provide enhanced gene expression but also revealed fewer epigenetic modifications than their cDNA counterparts [14, 92, 130]. Taking all the above data into consideration, we conclude that the usage of large genomic transgenes displays a more tissue specific, endogenous, and physiologically regulated gene expression pattern as they are controlled by native regulatory elements (Fig. 2). Henceforth, using gDNA with non-coding elements can be a promising strategy with high potential of overcoming the voluminous obstacles that currently exist in gene therapy.

Fig. (2).

Schematic comparison of possible advantages using gDNA locus versus cDNA vector in gene therapy.

10. GENOME EDITING APPROACHES IN GENE THERAPY

Over the past 10 years, genome editing approaches, such as clustered regularly interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR/Cas9) [131, 132] and base editing [133], have been used to directly correct genetic mutations in the field of gene therapy. Genome editing is achieved by delivering the editing machinery in situ that adds, deletes, and corrects genes, especially for the treatment of cancers and dominant inherited diseases, of which there is currently no standard treatment or cure [134]. Nowadays, genome editing is widely practiced and has proven results in animal models and pre-clinical studies. Numerous genome editing tools have been recently studied such as meganculeases, zinc-finger nucleases (ZFNs) and transcription activation-like element nucleases (TALENs) and CRISPR/Cas9. Although these tools are mainly used to edit coding regions, there are currently attempts to apply them in non-coding elements of the genome as well. Researchers have generated knockout mouse model of 18 lncRNA to study the functional role of 18 IncRNAs [135] and also have inserted an exogenous DNA sequences to study lncRNA MALAT1 in human lung cancer [136]. CRISPR/Cas9 is a gene editing strategy, which offers new methodology to study non-coding elements of the genome and is vastly used in studies involving genome editing and regulating of miRNA and lncRNA [137, 138]. It has reported that cloning CRISPR/Cas9 constructs with single guide RNAs can cause alterations on the miRNA, targeting the biogenesis processing sites of miRNAs both in vivo and in vitro [139]. CRISPR-based systems have been used to study gain of function and loss of function and also to identify enhancers and promoters in non-coding DNA to augment transcription, thereby offering greater scope in understanding non-coding DNA and their therapeutic applications [140]. A recent study has shown that co-delivering AAV receptor and the Sas Cas9 system can increase AAV transduction and enhance in vivo genome editing strategy in animal model [141]. Several innovative approaches to CRISPR-based technologies continue to emerge offer new ideas to study non-coding DNA and its therapeutic applications [140]. Concerns with use gene editing include the possibility of inducing unintended off-target mutations in the genome, tissue-specific targeting, distribution of the vector, pre-existing immunity of the host against engineered nucleases (immunogenicity) and biocompatibility of the carrier [142–145]. Compared with genome editing, using whole genome locus does not have off-target effects and the lesser immune response which is seen when the cells are genetically modified or edited in vivo. Genome editing also has rekindled the debate of germ-line gene therapy because of the ethical issues where there are risks of the procedure and the possible moral implications [146, 147]. Therefore, we still need time to evaluate the risks and benefits of using genome editing technologies.

11. CHALLENGES IN USING NON-CODING ELEMENTS IN GENE THERAPY AND DILIVERY VECTORS

Although it is well known that complete gDNA with correct native regulatory regions ensures physiologically relevant expression, there is still a lack of methodological studies that can confirm and assist in incorporating non-coding elements in gene therapy when compared to protein coding regions. This lack of understanding of how non-coding elements shape the activity in transgene expression and their function regarding gene regulation has hindered the development of effective therapeutic strategies to maintain transgene activity in vivo. Another challenge is the interpretation of non-coding variants that differ in each individual, which makes the functional work-up strategies nearly impossible [148]. Many of these challenges are currently investigated and can be addressed using NGS and bioinformatics tools to avoid false positive results. Nevertheless, as discussed previously the complex and intricate architecture of non-coding elements require more critical studies using appropriate tools, which will strengthen the development and implementation of non-coding elements in therapeutics.

The most commonly used viral vectors include retroviruses, lentiviruses, and adeno-associated viruses. Although these vectors have been successfully used in delivery of transgenes in gene therapy, there are very few vectors that can deliver large gDNA and non-coding elements. Epstein–Barr virus (EBV) is a relatively promising vector to deliver transgenes for gene therapy. It has been reported that EBV episomes can carry up to 660kb of gDNA and show persistent long term expressionin vivo and in vitro [114, 149]. The main advantage of EBV is its large transgene capacity and extrachromosomal persistence, making it promising vector for delivery of gDNA in gene [150, 151]. Researchers have used EBV for gene delivery for hemophilia B, some acquired diseases, and some types of cancer [152]. However, the major shortcomings of using EBC is its oncogenic potential, raising safety concerns about causing malignancies in humans [153, 154].

Artificial chromosomes (AC) are another common type of vector that have the capability to carry large gDNA. The commonly used ACs are human artificial chromosomes (HAC), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs). AC mediated gene delivery has the advantage of stable, low copy numbers, and delivery of large fragments of gDNA. These vectors have been used in delivery of gDNA with its regulatory elements and are studied in vitro and in vivo for stability and therapeutic efficiency [155–159]. Disadvantages include difficulty in making bacterial constructs, off target effects, immunogenicity risk, low transduction efficiency, and the fragility of the constructs. Those challenges will have to be overcome before they can be used in humans.

12. NANOPARTICLES IN NON-CODING ELEMENTS BASED GENE THERAPY

Recent advancements in genomics combined with rapid progression in nanotechnology have enhanced the enormous potential of gene therapy applications for many diseases, attracting worldwide attention. Using nanoparticles for gene delivery gained importance because of their smaller size, high surface to volume ratio, biocompatible properties like tunable hydrophobicity and hydrophilicity, low immune response, multiple functionalization, capability to go through surface modifications (chemically crosslinked structure), encapsulation efficiency and easy, inexpensive synthesis in large-scale, making them a promising candidate in gene therapy [160–162]. Nano delivery system can generally be divided into lipid-based nanoparticles such as liposomes, solidlipid particles, micelles and niosomes; polymeric nanoparticles such as chitosan and atelocollagen; dendrimers; inorganic nanoparticles such as nanotubes, metal-based nanoparticles, quantum dots, and nanogels; and silica nanoparticles [163–165]. They are used to deliver small molecules and also various bio macromolecules such as peptides, proteins, RNAs, plasmid DNA, and synthetic oligo deoxynucleotides. For the past few decades, various types of nanoparticles have shown targeted delivery of ncRNAs for diagnosis and therapy of various diseases including cancer cardiovascular and neurological diseases, making them a very attractive and promising option for gene therapy. Innovative RNA based therapeutics utilizing nanoparticle for delivery have shown great success in unravelling the existence of intricate hidden networks among various ncRNAs and their role in diseases like cancer.

One of the many advantages of nanoparticles is that some of them can deliver large DNA into cells without compromising their activity, safety, or quality. Our previous expertise in nanoparticles led to the identification of a complementary approach that can overcome various shortcomings in gene delivery previously discussed here were eliminated by using CK30PEG that can deliver large size gDNA loci to target the cell via a charge-driven self-assembly strategy [14, 166–171]. These nanoparticles can circumvent the size limitations and side effects of conventional delivery vehicles and deliver a 15–16kb full-length mouse and human rhodopsin gDNAs, including endogenous promoters, all introns, and flanking regulatory sequences into the rhodopsin knockout mouse (RKO) eyes in vivo. An interesting study that compared the effect of nanoparticle delivered plasmid cDNA to nanoparticle delivered gDNA counterpart driven by ubiquitous promoters showcased persistent rhodopsin gene expression and phenotypic improvement for up to 5 months post-injection [172]. We also demonstrated the importance of nanoparticle driven system of delivery of gDNA into an autosomal dominant retinitis pigmentosa (adRP) of the mutant rhodopsin P23H^+/− mouse model (unpublished data) to overcome the deleterious overexpression and under-expression of rhodopsin in photoreceptor cells. We are testing our hypothesis by using the gene’s own DNA sequence for transgene expression is superior to the artificial constructs currently in use since they use abbreviated promoters and lack endogenous introns and flanking sequences. We envision a period of utilizing large gDNA loci combined with nanotechnology, pinning great hopes on the arena of gene therapy.

13. IMPORTANCE OF NGS IN NON-CODING ELEMENTS BASED GENE THERAPY

Various experimental studies have validated the importance of exons, deep intronic, and large structural variants, but there is less evidence in identifying and associating the non-coding regions to diseases. GWAS are a recently booming area for studying most non-coding regions with vital regulatory functions in the mammalian genome, which offer invaluable information to analyze genomic regions to study localization of variants with regulatory activity in a particular tissue [173]. NGS technologies are now widely used in whole exome sequencing (WES), whole genome sequencing (WGS), and RNA sequencing (RNA-Seq) [174–176]. The NGS platform permits rapid and accurate sequencing of many genes in an individual for early detection of various complex life-threatening cancers, genetic diseases, heart diseases, infectious diseases, and immune disorders [174, 177]. It makes identifying genetic aberrations in various diseases easier and is demonstrated to be a competent tool for analyzing the role of non-coding DNA and RNA. Currently WGS is the most extensively used, as it can cover the entire genome that includes promoters, 5’UTR, 3’UTR, intragenic, intergenic segments, and other potential non-coding functional regions [178]. Ideally, it can be useful in delineating the functions of certain unrecognized genes in rare genetic disorders, making it an effective diagnostic tool in clinical practice, particularly in the field of single gene disorders [179]. Many scientific studies have used high-throughput sequencing methods such as ChIP-seq and chromosome conformation capture in combination with computational tools to analyze the functional role played by non-coding regions and their variants in the human genome. NGS data from a cohort of children having specific language impairment (SLI) was used to identify and validate the functionality of non-coding variants in this neurodevelopmental disorder [180]. This study also established the importance of non-coding 3′UTR variants in wider groups of neuropsychiatric disorders including schizophrenia (SCZ), autism spectrum disorder, and bipolar disorder, revealing the importance of these non-coding regions of a genome. Many reports from NGS data propose the existence of a major connection between various non-coding RNAs and neurodegenerative diseases like Alzheimer’s and Hereditary Cerebellar Ataxias (HCAs), and their essential role played in neurodegeneration-related processes [181, 182]. A recent study used WGS data in discovering non-coding driver mutations in 78 genes on a glioblastoma cohort previously associated with this disease [183]. It was also used as a diagnostic tool for a small family with inherited retinal degeneration and validated non-coding pathogenic variants involved in cone dysfunction syndrome [184]. In addition, an interesting report based on NGS data explained the significance of long neglected lncRNA genes in cardiovascular diseases, emphasizing the clinical implications of NGS in gene therapy [185–193]. There are around 304 studies reported in the https://clinicaltrials.gov/ website, which use miRNAs in clinical applications. Amongst which, phase I clinical trial NCT02369198, based on the administration of TargomiRs as 2nd or 3rd line treatment using nanoparticles for delivery for patients with recurrent malignant pleural mesothelioma and NSCLC is noteworthy. In addition, 13 studies are based on using lncRNAs (NCT02641847 phase I/II, NCT02221999 phase II/III and NCT03000764 - phase not applicable-) [194]. There are also studies that use lncRNAs in human trials to reduce tumor size by intra tumoral injection of targeting lncRNAS, where gene therapy is applied for delivery to specific cells [195].

Looking at all the above data, we can conclude that NGS has empowered scientists by providing abundant quantitative and qualitative data of the non-coding elements in the genome, which was ambiguous until recent years. This also adds to the need for more detailed analysis and interpretation of the available data to explain the functionality of non-coding elements and the advantages of their presence in full-length genomic loci. Therefore, we believe that the demand for NGS based gene therapy will the pave way for optimistic advancements in personalized therapy against actionable gene mutations. Exploiting NGS to study the impact of non-coding elements in gene expression and to validate whether they contribute in producing large expected changes in the phenotype will solve the puzzle which has been left incomplete for decades since the development of gene therapy.

CONCLUSION AND FUTURE PROSPECTIVES

Gene therapy is considered a potential revolution in medicine as it aims to treat or eliminate the cause of disease, whereas most current drugs address only the symptoms. It is a radical approach that offers superior targeting and persistent duration of action, facilitating longer biological effects that are more efficient and better localized to appropriate cells. A few decades ago, we witnessed many setbacks in the field of clinical gene therapy, but now it has attained success finding its way into the market. The first FDA approved gene therapy drugs, and Luxturna (voretigene neparvovec) in 2017 and Zolgensma (onasemnogene abeparvovec) in 2019 have created great hopes in the field of gene therapy. Developing strategies to meet off-target effect and extended gene expression with higher therapeutic efficiency promises a new paradigm in treating various diseases, ranging from genetic diseases to neurological disorders, cancer, and many infectious diseases. With the advances in NGS combined with emerging technologies of targeted therapy, we can identify and overcome more specific epigenetic alterations and uncover many key biological functions of non-coding segments in diseases, and their importance in gene therapy. We envision a time where we can overcome all the drawbacks that currently exist in gene therapy using NGS, pinning great hopes on the arena of gene therapy. The findings will take gene therapy one step further by revealing the potential to overcome several hurdles, including extended physiological stability and more therapeutic efficiency in treating genetic diseases.

Despite all these advances and success in gene therapy, further extensive experiments are needed to achieve unmitigated therapeutic efficiency in using a gDNA locus (with its regulatory elements) for treating diseases. We predict that using gDNA as the transgene will solve many unanswered queries in identifying the various epigenetic modifications, genetic source of diseases, appropriate drugs, and understanding of the pharmacological effects of these segments. Although gene therapy has many hurdles ahead, they are all surmountable if there is continuous effort from investigators in basic sciences to make scientific revolutions in the treatment of various existing and new diseases that might surface in the future.

LIST OF ABBREVIATIONS

AAV

Adeno-associated virus

cDNA

Complementary DNA

circRNA

Circular RNA

CRE

Cis-regulating element

CRISPR/Cas9

Clustered regularly interspaced short palindromic repeat-associated nuclease Cas9

ENCODE

Encyclopedia of DNA elements

FS

Flanking sequence

gDNA

Full-length genomic DNA loci

GWAS

Genome-wide association studies

lincRNA

Linear RNA

lncRNA

Long non-coding RNA

miRNA

Micro RNA

ncRNA

Non-coding RNA

NGS

Next generation sequencing

PR

Photoreceptor

RKO

Rhodopsin knockout mouse

RNA-seq

RNA sequencing

RP

Retinitis pigmentosa

siRNA

Small-interfering RNA

STRs

Short tandem repeats

WES

Whole exome sequencing

WGS

Whole genome sequencing