title: Management Genome Surgery for Sight: Advancements in Editing Technologies for Inherited Retinal Disorders- A Review
title: Gene Editing
titleType: Subtitle
title: CRISPR-Cas9
titleType: Subtitle
title: Viral vectors
titleType: Subtitle
title: Genetic Technology
titleType: Subtitle
title: siRNA
titleType: Subtitle
Creators: name: Atta Muhammad, Sumaira
name: Khaliq, Hafiz Muhammad Haseeb
name: Ashraf, Maria
Publisher: name: Zenodo
Year: 2023

description: INTRODUCTION: The neuroretina is a membrane located at the back of the eye that is sensitive to light. It is equipped with specialized sensory capabilities that allow it to capture light through photoreceptors, convert it into electric signals via phototransduction, and the signals need to be processed into an image at the central nervous system (CNS) level [1].Genome editing is a potent technique that has completely changed how we treat inherited retinal diseases including macular degeneration and retinitis pigmentosa. The functioning of the retina, the light-sensitive tissue at the back of the eye, is disrupted by a set of hereditary illnesses known as inherited retinal disorders [1, 2]. Mutations in the genes vital to the healthy growth and maintenance of the retina are the root cause of various illnesses. There are numerous varieties of inherited retinal illnesses, each with its own specific set of symptoms and course of development. The most prevalent ones include Stargardt disease, Leber congenital amaurosis and retinitis pigmentosa [3]. These disorders may result in a variety of visual abnormalities, such as blurred vision in dim light, loss of peripheral vision, and even total blindness. The nature of the condition and the age at which it is identified can both affect how severe the impairment is. Unfortunately, inherited retinal illnesses are currently incurable. Only controlling symptoms and halting the disease's course are available as treatments. For individuals with these illnesses, current developments in gene and cell therapies are providing fresh hope. Researchers are creating new medicines that can target the disease's underlying genetics by comprehending the genetics of inherited retinal illnesses. This includes employing cell treatment to replace unhealthy retinal cells with healthy ones or using gene therapy to replace unhealthy genes. Significant advancements in the treatment of hereditary retinal illnesses could result from these medicines, and continuous research is moving us closer to discovering a cure for these crippling diseases [4, 5]. 

Inherited retinal disorders are a group of rare genetic diseases that cause gradual vision loss and blindness. These disorders affect millions of people worldwide, and unfortunately, there is no known cure for them. However, recent advances in gene therapy offer hope for treating inherited retinal disorders [5]. Gene therapy involves delivering healthy genes to the cells in the affected part of the retina. These genes then produce functional proteins that help the retina to function normally. Gene therapy has shown promise in clinical trials, with some patients showing significant improvement in their vision. The success of gene therapy in treating inherited retinal disorders has encouraged researchers to develop new gene therapies and test them in clinical trials. In recent years, the field of gene therapy has made significant strides, and scientists are optimistic that gene therapy could become a mainstream treatment for inherited retinal disorders Gene therapy's ability to target the genetic cause of a disease is one of its key advantages. By replacing the faulty genes responsible for inherited retinal disorders, gene therapy offers a targeted and personalized treatment option that could potentially reverse the effects of the disease [6]. Gene therapy provides a targeted and personalized therapeutic approach that may be able to repair the consequences of the disease by replacing the defective genes that are responsible for inherited retinal abnormalities. Genome editing can be accomplished by delivering the editing machinery directly to the target site, either in vitro or in vivo, enabling precise and targeted modification of genes [5].

The treatment of inherited retinal illnesses may be revolutionized through genome editing. Technology advancements like CRISPR-Cas9 have made it possible for scientists to directly alter the genetic code, giving patients suffering from these crippling diseases new hope. Genome editing in hereditary retinal illnesses has produced guiding principles to help researchers and physicians overcome these obstacles. Based on a thorough understanding of the underlying science, these guidelines offer a foundation for safe and efficient gene therapy. One fundamental idea is the significance of ensuring that gene therapy is directed at the appropriate eye cells. The retina can be specifically targeted by particular viral vectors, or the required cells can be precisely located and targeted utilizing cutting-edge imaging methods. A second driving concept is the requirement for a thorough evaluation of the risks and advantages of gene therapy. Although inherited retinal problems may be cured with genome editing, there are hazards involved with any intrusive medical operation. Before deciding to get treatment, patients must be fully informed about the risks and advantages of gene therapy [7].
description: ABSTRACT: Inherited retinal disorders (IRDs) refer to a group of genetic diseases that are inherited and gradually lead to vision loss and blindness, affecting a significant number of people worldwide. Recent advancements in gene therapy and genome editing have opened up new possibilities for treating these conditions. IRDs are mainly caused by mutations in genes that are crucial to the functioning of the retina, leading to the impairment and eventual death of photoreceptor cells. Common examples of IRDs include Stargardt disease, Leber congenital amaurosis, and retinitis pigmentosa, with varying degrees of visual impairment. Traditional therapeutic methods aim to manage symptoms, but emerging strategies are bringing hope for better outcomes. Gene therapy, using viral vectors like AAVs, can introduce healthy genes into affected retinal cells, potentially restoring their function. Genome editing techniques like CRISPR-Cas9 can precisely alter or substitute faulty genes. Overcoming the challenges of effective delivery and sustained expression is key for widespread therapeutic success. Antisense oligonucleotides (ASOs) have emerged as promising candidates for addressing IRDs. ASOs can correct splicing defects or suppress the expression of mutated genes, resulting in improved retinal function. Continued advancements in ASO technology are expected to enhance their safety and effectiveness in tackling various genetic mutations linked to IRDs.Animal models and retinal organoids aid in studying retinal diseases. Adaptive optics help monitor photoreceptor health and treatment effectiveness.Developing long-lasting and comprehensive treatments for IRDs still has challenges. Research focuses on finding suitable gene delivery systems, ideal therapeutic dosage, and reducing immune responses. Collaborations across genetics, molecular biology, ophthalmology, and bioengineering fields is necessary to advance innovative treatments and bring hope to those affected by IRDs.
description: RETINAL DYSTROPHIES: Gene therapy for inherited retinal dystrophies (IRDs) has mainly concentrated on treating the eye. IRDs are degenerative illnesses caused by the absence of specific genes that encode normal proteins responsible for retinal cellular structures, phototransduction, or the visual cycle. The eye's anatomical and immunological features make it an ideal target for this type of therapy most hereditary eye disorders (IRDs), also known as retinal degenerations or dystrophies, result in permanent loss of vision and blindness. Gene mutations can cause the death of retinal photoreceptor cells, which contain photopigments such as opsin protein and a form of vitamin A called retina. These photopigments react to light, initiating the process of phototransduction at the photoreceptor outer segments. However, for this process to succeed, the retinal pigment epithelium (RPE) must be in good condition. The RPE is responsible for regenerating photochemically active retinal (vitamin A)-11-cis retinal, phagocytosing shed photoreceptors, and transporting nutrients and ions. Because photoreceptors use energy at high rates, they require a significant amount of energy Guidelines have been developed to aid researchers and physicians in addressing hereditary retinal illnesses through gene therapy. These guidelines, based on a comprehensive understanding of the science involved, serve as a basis for safe and effective treatment. A key principle is the importance of targeting the correct eye cells with the gene therapy. This can be achieved through the use of specific viral vectors or advanced imaging techniques for precise cell location and targeting. Another guiding principle is the need for thorough evaluation of the therapy's safety and efficacy [9]. A genetically and phenotypically diverse set of conditions known as inherited retinal degenerative diseases (IRDs) damage photoreceptor cells' ability to function. Significant visual impairment or complete blindness at a young age is a major cause of legal blindness among working-age adults. Unfortunately, the limited therapeutic options are due to the nature of neurodegenerative illnesses [10]. The pathophysiology and fundamental mechanisms of IRDs must be understood in order to find new therapeutic targets and demonstrate the efficacy of proposed treatment plans. The mouse has been the most often used mammalian animal model. The ability to apply research to the human condition is severely constrained by variations in the mix of retinal cell types, in the gene expression specific to different cell types, and in the macroscopic organization of the retina. Researchers have used induced pluripotent stem cells (iPSCs) to create human retinal organoids. This tool is useful for researching IRDs that don't have animal models or have human mutations that can't be replicated in them. Advances in molecular genetics have made it possible to identify the genes responsible for mutations in enzymes and retinoid-binding proteins in diseases such as Leber congenital amaurosis (LCA), Stargardt disease (STGD), cone dystrophy (COD), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP) [7].  

Since IRDs lead to the decline of photoreceptors and visual impairment, imaging living eyes noninvasively can shed light on the structure of rods and cones as the disease progresses and how it responds to potential treatments. It's unfortunate that most clinical imaging techniques cannot visualize individual photoreceptors at the cellular level due to the lack of necessary resolution. However, adaptive optics is a method that can measure aberrations in the light leaving the eye. Adaptive optics scanning laser ophthalmoscopy (AOSLO) captures confocal light wave-guided by photoreceptors with intact inner and outer segments. These systems also have the potential to be sensitive, objective outcome measures of safety and efficacy in clinical trials for IRD patients [7, 12].

GENE DELIVERY SYSTEM: 

In recent times, a diverse array of both viral and nonviral vectors has undergone rigorous evaluation for their potential to effectively transduce the critical retinal pigment epithelium (RPE) and photoreceptor cells, which are primarily affected in retinal degenerative diseases. The selection of an appropriate vector is pivotal in the context of gene therapy endeavors (summarised in Table 1-see next page). When addressing retinal dystrophy, it becomes imperative to achieve sustained gene correction, necessitating prolonged transgene expression with minimal risk of eliciting immunogenic responses. Furthermore, avoiding integration into the host genome is a priority to mitigate potential adverse events associated with insertional mutagenesis. Beyond these criteria, the optimal vector should ideally display cell specificity, accommodate substantial cloning capacity, exhibit low toxicity, and be amenable to large-scale and high-purity production. The use of viral vectors like adenovirus, lentivirus, and adeno-associated virus (AAV) is common for delivering therapeutic genes to diseased retinas and restoring targeted cell function [13].

VIRAL VECTORS: Adenoviruses are widely used for gene therapy as they can effectively transfer genes to different types of cells and ensure strong gene expression even during the cellular cycle stage. However, they may face limitations when it comes to photoreceptor transduction, except during specific developmental phases or stages of degeneration [14]. It's possible that the complexity of the fully developed mouse retina's structure may hinder the successful transfer of genetic material using viral vectors. Interestingly, adenoviral vectors have shown potential in addressing issues with the retinal pigment epithelium (RPE). Research has shown that the administration of recombinant adenovirus expressing the MERTK gene in RCS rats has led to improvements in photoreceptor function and an increase in photoreceptor numbers [15]. Adenoviral vectors have a unique ability to hold large genetic inserts of up to 48 kb. However, there are concerns regarding safety and the limited duration of gene expression. This is caused by the activation of specific viral genes in the vector backbone, which may trigger an immune response. As a result, this response can cause temporary transgenic expression and long-term toxicity [16]. When prolonged transgenic expression is necessary, adenoviral vectors may not be the best option. Research is focused on reducing viral genes in recombinant virions to enhance safety and efficacy [17]. The first-generation vectors, with deletions within the E1 and E3 regions, are rendered replication-deficient and capable of housing DNA inserts of up to 7.5 kb [18]. Further deletions or mutations in viral regions have resulted in increased safety and effectiveness in subsequent generations [19]. New and improved helper-dependent adenoviral vectors do not contain viral coding sequences but maintain high efficiency of in vivo transduction and transgene expression, like first-generation adenoviral vectors. With these vectors, there is high-level and sustained transgene expression without causing chronic toxicity [20]. Challenges have arisen due to helper virus contamination, vector instability, and the emergence of replication-competent adenoviruses [21].

LENTIVIRUSES (LVs): Lentiviruses are retroviruses with reverse transcriptase activity. They can integrate proviral DNA into host cell chromosomes. This property distinguishes LVs from traditional retroviruses, allowing them to effectively transduce both dividing and nondividing cells, thereby facilitating stable transgene expression [22]. However, the propensity for integration-associated risks has spurred the exploration of non-integrating vectors. Intriguingly, in vivo experiments employing non-integrating lentiviral vectors have demonstrated sustained transgene expression within post-mitotic tissues such as the retinal pigment epithelium (RPE). The gene cargo capacity of lentiviral vectors ranges between approximately 8–10 kb, rendering them capable of transducing RPE cells along with differentiated photoreceptors [23].

ADENO-ASSOCIATED VIRUSES (AAVS):  AAV-based vectors are a promising option for treating inherited retinal diseases (IRDs) due to their effectiveness and ability to provide stable gene transfer. Despite their limited packaging capacity, recent research has been focused on improving the transfer capacity of AAV vectors. These vectors have the capability to infect both dividing and non-dividing cells and have a wide range of tropism, which depends on the specific serotype used [25]. Pseudo typed vectors have played a crucial role in enabling cell-specific targeting strategies [26, 27]. AAV-based therapies have demonstrated notable successes across animal models and clinical trials [28, 29].

NONVIRAL GENE TRANSFER: Nonviral approaches, encompassing naked DNA, nanoparticles (NPs), and liposomes, have been explored as viable alternatives to viral vectors. While nonviral methods are notably cost-effective and practical, their efficacy and potential for sustained gene expression often lag behind that of viral vectors. Among these, the administration of naked plasmid DNA via electroporation has demonstrated promise, albeit transgene expression tends to be transient [30]. Nanoparticles, particularly DNA–gold NPs, present an intriguing avenue, although challenges concerning internalization routes and the sustainability of expression have emerged [31]. Liposome-mediated transfection, while presenting introducing genes, is generally characterized by lower efficiency compared to viral vectors [32]. 

GENE REPLACEMENT: The gene replacement strategy has emerged as a particularly effective approach for addressing inherited retinal diseases (IRDs). This strategy involves introducing functional copies of defective genes to restore cellular function and mitigate degeneration. Notable successes have been recorded in animal models through gene replacement therapy [33]. These breakthroughs are particularly significant as they open avenues for treating a diverse range of IRDs by providing functional alternatives to malfunctioning genes, thereby restoring cellular functionality and arresting the progression of degeneration.

GENE SILENCING: Autosomal dominant retinitis pigmentosa (adRP) refers to a group of inherited retinal disorders where the mutated RHO gene, responsible for encoding rhodopsin, accounts for approximately 30-40% of cases [34]. This type of adRP is characterized by a diverse range of mutations, a common feature observed in many inherited diseases. The production of abnormal proteins due to mutations causes RHO-adRP, disrupting the functioning of the wild-type protein and leading to cellular toxicity.The therapeutic approach for RHO-adRP focuses on repairing or silencing the mutated gene to mitigate the disease progression [35].

There are two proposed strategies for silencing mRNA in this condition. The first strategy involves using specific inhibitors that target the mutated mRNA, causing its degradation. This is necessary for maintaining the function of photoreceptor cells by allowing the normal allele to be expressed [36]. The second strategy is to inhibit the transcripts of both alleles and replace them with an unaltered wild-type form, regardless of mutation status [37]. Antisense inhibitors, including ribozymes, small RNA inhibitors (siRNA), and antisense oligonucleotides can be employed to implement both approaches.

RIBOZYME: Silencing specific genes associated with RD using ribozymes can help mitigate the progression of the disease. Ribozymes can be designed to target and degrade the mutated mRNA responsible for the pathogenesis of the disease [38]. By cleaving the RNA molecule in a sequence-specific manner, ribozymes can selectively inhibit the expression of the mutated gene, allowing the normal allele to be expressed [38]. RNA molecules called ribozymes can act as catalysts. Hairpin and hammer ribozymes are commonly used to selectively inhibit gene expression due to their precise size. Both types of ribozymes rely on proper folding to cleave a particular phosphodiester bond through a transesterification reaction [39]. Metal ions such as magnesium, manganese, calcium, and cobalt ions can trigger the formation of distinct folded structures. Scientists have researched various approaches, including mutations and mutation-independent techniques, to enhance the potential of ribozymes in vitro. A study demonstrated that an aib-driven ribozyme effectively combated p23h rat model mutations [1]. Gorbatyuk et al. found reduced rhodopsin mRNA and decreased b wave amplitudes in a mouse model (Rho+/−) following AAV-mediated ribozyme injection at P6 [40]. Researchers conducted a study using a more powerful ribozyme on a rat model with a mix of a transgenic mouse mutant allele and a wild-type rat allele. This ribozyme was effective in dogs, mice, and humans but not in rats. During the experiment, the unaffected mouse gene replicated the effect of the replacement gene. After around a month of injection, the treated eyes showed a significant preservation of about 50% of the a and b wave amplitudes of the scotopic ERG. However, the ERG response in the injected eyes continued to decline at a similar rate to the non-injected eyes. This shows that ribozymes can restrict gene expression by targeting mRNA in the retina in vivo. However, the ribozyme recognition sequence is widely present in the genome, leading to increased off-target effects [41]. Using allele-independent RNA inhibitors is a more convenient option when combating mutations in the same gene. However, for optimal treatment, it is important to supply both the wild-type protein and RNA inhibitors together. Packing both sequences into one AAV is not possible due to their large size, which means that delivering them will be a crucial consideration.

siRNA: In vivo experiments have demonstrated promising results with the use of ribozymes. However, a more effective and enduring approach involves the use of small interfering RNA (siRNA) technology for RNA inhibition. This method targets and cleaves complementary mRNA by utilizing sequence-specific siRNA [42]. The enzyme Dicer produces siRNA fragments, which are 21-23 nucleotides long, from long dsRNA. Alternatively, synthetic siRNA can be introduced into the cell directly [44]. Getting siRNAs to the back of the eye can be difficult, and the best way to do it is with an injection into the eye. Sometimes more than one injection is necessary because the siRNA can break down, which can cause problems like cataracts, retinal detachment, bleeding in the eye, and inflammation of the eye [45]. In order to tackle these obstacles, scientists have investigated the possibility of enclosing siRNAs within nanocarriers that are nonviral [46]. The negative charge of glycosaminoglycans can restrict their movement and cause clumping. While polyethylene glycol can stop clumping, it can also hinder the way cells take up and release substances. A promising solution is to use the Transit-TKO transfection reagent as a carrier for delivering siRNA into the eye.This method has shown success in mice after being injected into the retina [47]. There are researchers who suggest the use of siRNA to silence rhodopsin mRNAs as a potential method to decrease the rate of retinal degeneration in patients with adRP. They propose two distinct approaches. The first approach concentrates on the mutated alleles, halting their transcription while enabling the normal expression of wild-type alleles [17]. However, this approach was not effective in preventing retinal degeneration in a rat model. [18Additionally, due to the extensive range of rhodopsin gene mutations, creating personalized therapies for each mutation would require significant time and resources. Another approach is to use siRNAs to inhibit both the mutant and wild-type alleles of the rhodopsin gene, while introducing a modified rhodopsin gene that is resistant to inhibition.[48]. Studies have discovered a hopeful method to impede rhodopsin RNA and substitute it with fresh genes in mice, which has also proven effective in decreasing retinal degeneration in mice with the P23H mutation - a condition associated with human RP. Through a combination of rhodopsin siRNA that is not dependent on mutations and rhodopsin RNA replacement, mice with this mutation have achieved long-term repair of their retinal structure and function.[49]. SiRNAs have been combined with gene replacement technology to suppress RHO and RDS genes, among others, for improved RP therapies [50]. Studies show that using RNA inhibition and gene substitution can improve photoreceptors after treating animal models of adrp.

 

ANTISENSE OLIGONUCLEOTIDES (ASO): This therapy has the potential to treat various types of retinal dystrophies, which are disorders characterized by progressive dege neration of retinal cells and subsequent vision loss. Antisense oligonucleotides (ASOs) are synthetic DNA or RNA segments can be designed to target specific genetic mutations or aberrant gene expression associated with retinal dystrophies [51]. Examples include DMD-associated retinal dystrophy, a genetic disorder that affects muscle function but can also lead to vision impairment [52]. ASOs can restore the reading frame and potentially produce a functional dystrophin protein, alleviating retinal dystrophy symptoms. RPGR-associated retinitis pigmentosa (X-linked RP) is a severe form of retinal dystrophy caused by mutations in the RPGR gene [53]. ASOs are being developed to target specific mutations in the RPGR gene, aiming to correct splicing defects and restore proper protein function.

Usher Syndrome Type 2A, a genetic disorder involving hearing loss and retinal degeneration, is caused by mutations in the USH2A gene [54]. ASO-based therapies for retinal dystrophies are at different stages of development, ranging from preclinical research to clinical trials. The success of these therapies depends on factors such as accurate targeting of genetic mutations, optimal design of ASOs, efficient delivery methods, and careful evaluation of their effects on preserving or improving retinal function [51]. AON-based therapy has advantages over gene enhancement therapy. AON molecules can be directly injected to the retina with no harmful side effects on the immune system via intravitreal or subretinal injections [55,56]. Two recent studies have confirmed that retinal AON administration is safe and effective. One study showed an increase in exon targeting without any negative effects, while the other used AONs to break down the RHO mutant allele in rats with autosomal dominant RP [57,58].

CRISPER Cas9 SYSTEM: GENOME EDITING: The CRISPR/Cas9 system is a novel approach for gene editing that uses single-guide RNA (sgRNA) to deliver the nuclease Cas9 to target sites, resulting in double-stranded DNA breaks (DSB). Cas nucleases identify DNA point breaks using two sequences: the RNA guide and the neighbouring protospacer motif (PAM). The CRISPR/Cas system may target any DNA sequence, with Cas9 from Streptomyces pyogenes (SpCas9) being the most often utilised nuclease. Once in cells, the target sequence's double strand can be severed and repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR) [59-61]. NHEJ is a genome editing mechanism that can result in the addition or removal of genetic material from DSBs. This often leads to the creation of dysfunctional proteins due to premature stop codons. If an RNA guide targets one or both alleles, it can be used to block mutant alleles. However, if Cas9 recognizes both alleles, integration of the wild-type gene is necessary. NHEJ is commonly used for gene deletion [62-64]. Although HDR is a high-fidelity process, it is regarded as inefficient since it depends on cell division and can only modify the genome of germline or proliferating cells as shown in many alternative malignancies such as thyroid Tumorigenesis [65-70]. It either employs single-stranded donor oligonucleotides sequences with the Cas9 nuclease as a template or sister chromatids of the homologous chromosome. When cells are dividing in the S and G2 stages, HDR occurs less frequently than NHEJ.

 

The Cas9-mediated HDR pathway, however, is seldom used in postnatal tissues and cells since the majority of cells in postnatal tissues in mammals lose their ability for proliferation as they develop. For the purpose of treating RP illness, created a genome-based HDR-based editing approach to fix a genetic mutation in rd1 postnatal mice retinas. They used HDR to develop a novel Cas9/RecA system that fixed the Pde6b mutation and stopped in vivo photoreceptor degeneration. Compared to the NHEJ pathway, which has poor high-fidelity alterations and error-prone repair, this strategy provides a wider range of applications in the treatment of diseases. The exact insertion of desired genetic engineering is made possible by HDR-based genome editing. Since HDR is less effective than NHEJ, several methods for targeted sequence insertion employing modified nucleases have been developed. Potentially, RP and other hereditary disorders caused by genetic abnormalities might be treated using the Cas9/RecA system [71]. The promise of genetic editing as a treatment for hereditary illnesses will be fulfilled by the ongoing development of this technology to increase the safety and effectiveness of genetic editing as shown in Figure above.  Schematic representation of gene editing technologies in the treatment of retinal dystrophies.
description:  

CONCLUSION: Recent advances in genome editing techniques have revolutionized the approach to treating inherited retinal disorders (IRDs) like macular degeneration and retinitis pigmentosa, offering hope for effective treatments through gene therapy. While traditional treatments focus on symptom management, gene therapy's ability to address underlying genetic causes represents a paradigm shift. The strategic selection of gene delivery systems is crucial for efficient and sustained transgene expression. With gene therapies moving closer to becoming mainstream treatments, there is renewed hope for individuals affected by IRDs, potentially reversing the devastating effects of these disorders. 

Gene therapy for inherited retinal disorders (IRDs) holds promise with advancements in genome editing, such as base and prime editing. Understanding the DNA repair status in retinal cells is crucial for precise gene therapy. Blocking NHEJ proteins can enhance HDR activity, though more research is necessary. Safety is key, preventing off-target toxicity and immune responses in retinal cells. AAV vectors show promise, but Cas9 RNP delivery reduces off-target effects. Whole exome sequencing is a comprehensive approach for post-ex vivo gene correction.
description: REFERENCES

 

1.          Ziccardi, L., et al., Gene therapy in retinal dystrophies. International journal of molecular sciences, 2019. 20(22): p. 5722.

2.         Chapman, J.E., D. Gillum, and S. Kiani, Approaches to reduce CRISPR off-target effects for safer genome editing. Applied Biosafety, 2017. 22(1): p. 7-13.

3.         Bainbridge, J.W., et al., Effect of gene therapy on visual function in Leber's congenital amaurosis. New England Journal of Medicine, 2008. 358(21): p. 2231-2239.

4.         Rodrigues, G.A., et al., Pharmaceutical development of AAV-based gene therapy products for the eye. Pharmaceutical research, 2019. 36: p. 1-20.

5.         Li, H., et al., Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal transduction and targeted therapy, 2020. 5(1): p. 1.

6.         Gilliland, D.G., Molecular genetics of human leukemia. Leukemia (08876924), 1998. 12.

7.         Zhang, H., et al., MAP4Ks inhibition promotes retinal neuron regeneration from Müller glia in adult mice. NPJ Regenerative Medicine, 2023. 8(1): p. 36.

8.         Scholl, H.P., et al., Emerging therapies for inherited retinal degeneration. Science translational medicine, 2016. 8(368): p. 368rv6-368rv6.

9.         Duncan, J.L., et al., Inherited retinal degenerations: current landscape and knowledge gaps. Translational vision science & technology, 2018. 7(4): p. 6-6.

10.       Cursiefen, C., et al., Unmet needs in ophthalmology: A European Vision Institute-consensus roadmap 2019–2025. Ophthalmic Research, 2019. 62(3): p. 123-133.

11.       Bujakowska, K.M., Q. Liu, and E.A. Pierce, Photoreceptor cilia and retinal ciliopathies. Cold Spring Harbor Perspectives in Biology, 2017. 9(10): p. a028274.

12.       Morgan, J.I., The fundus photo has met its match: optical coherence tomography and adaptive optics ophthalmoscopy are here to stay. Ophthalmic and Physiological Optics, 2016. 36(3): p. 218-239.

13.       Zeng, Y., et al., RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave. Investigative ophthalmology & visual science, 2004. 45(9): p. 3279-3285.

14.       Li, T., et al., In vivo transfer of a reporter gene to the retina mediated by an adenoviral vector. Investigative ophthalmology & visual science, 1994. 35(5): p. 2543-2549.

15.       Vollrath, D., et al., Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proceedings of the National Academy of Sciences, 2001. 98(22): p. 12584-12589.

16.       Kafri, T., et al., Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression. Proceedings of the National Academy of Sciences, 1998. 95(19): p. 11377-11382.

17.       Verma, I.M. and M.D. Weitzman, Gene therapy: twenty-first century medicine. Annu. Rev. Biochem., 2005. 74: p. 711-738.

18.       Amalfitano, et.al., Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors. Proceedings of the National Academy of Sciences, 1996. 93(8): p. 3352-3356.

19.       Dudley, R.W., et al., Sustained improvement of muscle function after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector. Human gene therapy, 2004. 15(2): p. 145-156.

20.       Steinwaerder, D.S., C.A. Carlson, and A. Lieber, Generation of adenovirus vectors devoid of all viral genes by recombination between inverted repeats. Journal of virology, 1999. 73(11): p. 9303-9313.

21.       Greenberg, K.P., et al. Gene delivery to the retina using lentiviral vectors. in Retinal Degenerative Diseases. 2006. Springer.

22.       Yánez-Munoz, R.J., et al., Effective gene therapy with nonintegrating lentiviral vectors. Nature medicine, 2006. 12(3): p. 348-353.

23.       Cashman, S.M., et al., Intercellular trafficking of adenovirus-delivered HSV VP22 from the retinal pigment epithelium to the photoreceptors—implications for gene therapy. Molecular Therapy, 2002. 6(6): p. 813-823.

24.       Trapani, I., Adeno-associated viral vectors as a tool for large gene delivery to the retina. Genes, 2019. 10(4): p. 287.

25.       Surace, E.M. and A. Auricchio, Versatility of AAV vectors for retinal gene transfer. Vision research, 2008. 48(3): p. 353-359.

26.       Ferrari, F.K., et al., Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. Journal of virology, 1996. 70(5): p. 3227-3234.

27.       Thomas, C.E., et al., Rapid Uncoating of vector genomes is the key toefficient liver transduction with pseudotyped adeno-associated virusvectors. Journal of virology, 2004. 78(6): p. 3110-3122.

28.       Ali, R.R., et al., Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Nature genetics, 2000. 25(3): p. 306-310.

29.       Cai, X., S.M. Conley, and M.I. Naash, RPE65: role in the visual cycle, human retinal disease, and gene therapy. Ophthalmic genetics, 2009. 30(2): p. 57-62.

30.       Prud'homme, G.J., et al., Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases. Current Gene Therapy, 2006. 6(2): p. 243-273.

31.       Trigueros, S., et al., In vitro gene delivery in retinal pigment epithelium cells by plasmid DNA-wrapped gold nanoparticles. Genes, 2019. 10(4): p. 289.

32.       Azzam, T. and A.J. Domb, Current developments in gene transfection agents. Current drug delivery, 2004. 1(2): p. 165-193.

33.       Allocca, M., et al., Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. The Journal of clinical investigation, 2008. 118(5): p. 1955-1964.

34.       Massengill, M.T. and A.S. Lewin, Gene therapy for rhodopsin-associated autosomal dominant retinitis pigmentosa. International ophthalmology clinics, 2021. 61(4): p. 79.

35.       Meng, D., S.D. Ragi, and S.H. Tsang, Therapy in rhodopsin-mediated autosomal dominant retinitis pigmentosa. Molecular Therapy, 2020. 28(10): p. 2139-2149.

36.       Seyhan, A.A., RNAi: a potential new class of therapeutic for human genetic disease. Human genetics, 2011. 130(5): p. 583-605.

37.       Kiang, A.-S., et al., Toward a gene therapy for dominant disease: validation of an RNA interference-based mutation-independent approach. Molecular Therapy, 2005. 12(3): p. 555-561.

38.       Ku, C.A. and M.E. Pennesi. The new landscape of retinal gene therapy. in American Journal of Medical Genetics Part C: Seminars in Medical Genetics. 2020. Wiley Online Library.

39.       Alonso, D. and A. Mondragón, Mechanisms of catalytic RNA molecules. Biochemical Society Transactions, 2021. 49(4): p. 1529-1535.

40.       Sudharsan, R. and W.A. Beltran, Progress in gene therapy for rhodopsin autosomal dominant retinitis pigmentosa. Retinal Degenerative Diseases: Mechanisms and Experimental Therapy, 2019: p. 113-118.

41.       O'Reilly, M., et al., RNA interference-mediated suppression and replacement of human rhodopsin in vivo. Am J Hum Genet, 2007. 81(1): p. 127-35.

42.       Gorbatyuk, M., et al., Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme. Experimental Eye Research, 2007. 84(1): p. 44-52.

43.       Elbashir, S.M., W. Lendeckel, and T. Tuschl, RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 2001. 15(2): p. 188-200.

44.       Elbashir, S.M., et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411(6836): p. 494-498.

45.       Janoria, K.G., et al., Novel approaches to retinal drug delivery. Expert Opin Drug Deliv, 2007. 4(4): p. 371-88.

46.       Morales-Becerril, A., et al., Nanocarriers for delivery of siRNA as gene silencing mediator. Excli j, 2022. 21: p. 1028-1052.

47.       Turchinovich, A., G. Zoidl, and R. Dermietzel, Non-viral siRNA delivery into the mouse retina in vivo. BMC Ophthalmology, 2010. 10(1): p. 25.

48.       Kiang, A.-S., et al., Toward a Gene Therapy for Dominant Disease: Validation of an RNA Interference-Based Mutation-Independent Approach. Molecular Therapy, 2005. 12(3): p. 555-561.

49.       Mao, H., et al., Long-term rescue of retinal structure with a single adeno-associated viral vector in P23H RHO transgenic mice. Human gene therapy, 2012. 23(4): p. 356-366.

50.       Petrs-Silva, H., et al. Suppression of rds expression by siRNA and gene replacement strategies for gene therapy using rAAV vector. in Retinal Degenerative Diseases. 2012. Springer.

51.       Xue, K. and R.E. MacLaren, Antisense oligonucleotide therapeutics in clinical trials for the treatment of inherited retinal diseases. Expert Opinion on Investigational Drugs, 2020. 29(10): p. 1163-1170.

52.       Fortunato, F., M. Farnè, and A. Ferlini, The DMD gene and therapeutic approaches to restore dystrophin. Neuromuscular Disorders, 2021. 31(10): p. 1013-1020.

53.       Martinez-Fernandez de la Camara, et.al. Emerging gene therapy products for RPGR-associated X-linked retinitis pigmentosa. Expert Opinion on Emerging Drugs, 2022. 27(4): p. 431-443.

54.       Delmaghani, S. and A. El-Amraoui, The genetic and phenotypic landscapes of Usher syndrome: from disease mechanisms to a new classification. Human Genetics, 2022. 141(3-4): p. 709-735.

55.       Rowe-Rendleman, C.L., et al., Drug and gene delivery to the back of the eye: from bench to bedside. Investigative ophthalmology & visual science, 2014. 55(4): p. 2714-2730.

56.       Gérard, X., et al., Intravitreal injection of splice-switching oligonucleotides to manipulate splicing in retinal cells. Molecular Therapy-Nucleic Acids, 2015. 4.

57.       Murray, S.F., et al., Allele-specific inhibition of rhodopsin with an antisense oligonucleotide slows photoreceptor cell degeneration. Investigative ophthalmology & visual science, 2015. 56(11): p. 6362-6375.

58.       Garanto, A., et al., In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet, 2016. 25(12): p. 2552-2563.

59.       Komor, A.C., A.H. Badran, and D.R. Liu, CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell, 2017. 169(3): p. 559.

60.       Nelson, C.E., et al., In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science, 2016. 351(6271): p. 403-7.

61.       Yin, H., et al., Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature biotechnology, 2014. 32(6): p. 551-553.

62.       Zhou, J., et al., One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. International j. of biochem & cell biology, 2014. 46: p. 49-55.

63.       Wu, W.-H., et al., CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa: a brief methodology. Retinal Gene Therapy: Methods and Protocols, 2018: p. 191-205.

64.       Wu, Y., et al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell, 2013. 13(6): p. 659-662.

65.       Batool A, Khaliq HMH, Arshad A. Beyond the Gland: Thyroid Disorders Unraveled as a Distinctive Challenge in Health and Disease Management. Zenodo; 2022 Aug.

66.       Rana S, Khaliq HMH, Hina M, Arshad A. Comparative Study of Bacterial Rejection Properties of Polymer Membranes Fabricated with Silver and Titania Nanoparticles. Zenodo; 2023.

67.       Javed N,et.al. Identification of Tumorigenic Markers in Head and Neck Cancer Patients, Journal of Health and   Rehabilitation Research. 2023 Dec      25;3(2):808-16.

68.       Khaliq HMH, Batool A, Arshad A. The Heart of Tumorigenesis: Dissecting the c-Met/HGF Pathway in Cardiac Cancer. Zenodo; 2023 May.

69.       Shafique S, Haseeb HM, et.al. Investigating Entosis and Autophagy Dynamics in Diverse Tumor Microenvironments , Pak-Euro Journal of Medical and Life Sciences: Vol. 7, 3 (2023) Dec:219-226.

70.       Arshad A, et.al. (2015) Simultaneous Irradiation of Fibroblasts and Carcinoma Cells Repress the Secretion of Soluble Factors Able to Stimulate Carcinoma Cell Migration. PLoS ONE 10(1): e0115447.

71.       Cai, Y. and T. Cheng, In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway. 2019. 5(4): p. eaav3335.