Gene therapy in PIDs, hemoglobin, ocular, neurodegenerative, and hemophilia B disorders

1 Background

Molecular biology and biotechnology tools remained important elements in gene therapy. Gene editing/modification (replacement, insertion, and deletion) largely characterizes this field of biological sciences. The idea of gene therapy was implemented clinically about three decades ago as an alternative to the limitations of pharmacotherapy. Approximately 3,000 known clinical trials are on record (Table 1). Some limitations are associated with gene therapy; and hence, the need to improve on the current strategies. This has resulted in sophisticated tools using viral and nonviral vectors (Figure 1). Although most of the gene therapy studies are directed toward cancer worldwide (http://www.wiley.com/legacy/wileychi/genmed/clinical/), other areas of notable disease require the gene therapy approach; and these include primary immunodeficiency disorders (PIDs), hemoglobin, hemophilia B, ocular, and neurodegenerative disorders. Gene therapy encompasses the introduction of new therapeutic genes, as DNA segments, modification of existing genes, or the introduction of RNA into cells, with the aim of preventing, treating, or curing disorders and diseases in order to restore or add gene expression. Diseases such as diabetes, Parkinson’s disease, heart failure, cancers, and neurodegenerative and metabolic disorders have been well managed through gene therapy [1].

Figure 1

Viral and nonviral vectors used in gene therapy. Data sourced from: www.wiley.co.uk/genmed/clinical, 2018.

These strides are, however, not devoid of challenges that include recognizing the protein on the viral capsid by host immune system; delineating it as an antigen, though this has been managed through the development of elaborate technologies; and shielding the protein sites from eliciting inflammatory activities. Hence, this produces excellent safety profiles even for in vivo gene therapy [2]. A key advantage of this protocol is that adverse reactions and side effects are reduced to a feasible minimum, and it also provides room for the administration of the desired concentration. Therapeutic gene treatment also holds healthier potentials for use in managing diseases such as hemoglobinopathies, cancer immunotherapies, hemophilia B, ocular diseases, neurodegenerative diseases [3], immunological and metabolic disorders, and hematological diseases that have eluded cure, in resistance to the conventional treatment options [4,5]. Rapid improvements are recorded every year. Many phases of clinical and experimental trials are in the pipeline to improve on all the factors surrounding the anticipated accomplishments in gene therapy. It is, however, very pertinent to keep the medical and research community abreast of the most recent developments and redirections discussed under some of the systematic headings in the selected areas of PIDs, hemoglobin, ocular, neurodegenerative, and hemophilia B disorders as addressed in this study. We further briefly highlight the application of the more recent gene-editing tool, clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR-cas9), but will be discussed in greater detail in the future reviews.

2 The path to treating PIDs

Records have been documented for many monogenic disorders (inheritable disorders that are a result of a single-defective gene on the autosomes) that require treatment using gene therapy. These are caused by a mutation in a single gene. Examples include cystic fibrosis, sickle cell disease, Wiskott–Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Tay–Sachs disease, polycystic kidney disease, and PIDs, which comprise a class of rare, inheritable disorders of the immune system. A typical instance includes the severe combined immunodeficiency ([SCID] e.g., adenosine deaminase SCID [ADA-SCID] and IL-2 receptor X-linked severe combined immunodeficiency [SCID-X1]) [6]. The single most common strategy in dealing with these forms of diseases has been the genetic modification of hematopoietic stem cell (HSC), which has proven efficacious over the years. For example, HSC can be transplanted from a donor that has a similar and compatible HLA to a beneficiary (patient) to treat PID. Over 90% success has been recorded in such trials [7]. Substantial feasibility has been documented for the ex vivo delivery of a single gene into the isolated HSC, followed by transplant, with very well-established protocols. However, because of the complications associated with mismatch and incompatibility with some donors and their recipients, it is considered more expedient to modify the patient’s own HSC and readminister to the patient. Therefore, the donor is the recipient, and this factor rules out compatibility complications.

The phenotypic manifestation of the CGD is that the obviously mature phagocytes cannot carry out their function of killing the ingested microorganisms, potentially leading to serious inflammation. p22phox, gp91phox, p67phox, p47phox, and p40phox are all genes coding for the NADPH oxidase complex of the phagocytes, and it is in this set of genes that mutations occur, thereby impairing their function [8]. The catalytic subunit gp91phox coded by the cytochrome b (558) gene (CYBB) is located on the X-chromosome (X-CGD) and is usually affected (mutated) in most disease cases. Hence, treatment by delivering gp91phox as a transgene cures patients without major side effects. Prominent locations where ADA deficiency has been treated include the US, UK, and Italy, with commendable successes [9], though not 100% of the time. For example, in the case of some SCID-X1 patients treated by gene therapy, the enthusiasm was dampened as 2–6 years later an onset of acute T-cell lymphoblastic leukemia (T-ALL) was reported in 5 of 60 patients who received HSC as gene therapy in the past [10]. However, the situation was resolved or managed by the activation of LIN-11, Isl-1 and MEC-3 (LIM) domain only 2 (LMO2), a proto-oncogene, and a transcriptional cofactor capable of promoting the self-renewal of committed T cells [11]. The previous intended therapeutic use of gamma retroviral vectors (usually containing intact 50 long terminal repeats [LTRs]) produced severe adverse effects that were noticed in early implementation of gene therapy trials, calling for a search into the process of retroviral incorporation into human cell lines as well as primary human HSCs (CD34þ), which ultimately elucidated the need to search for better alternatives [12]. Even when using gene therapy methods, different outcomes have been recorded with different and distinct PIDs. This necessitates the establishment of protocols that consider the uniqueness of the different diseases, especially as they relate to vector design. This is actually dependent on the fundamental understanding of the molecular basis for interaction.

It is important to note that viral vector integration is actually an active process catalyzed by the tethering of the viral preintegration complex to the open chromatin regions in the genome of the host cell characterized by DNaseI hypersensitive sites as well as epigenetic marks [12]. Considering all the previous points so far, vector–chromatin interaction and its consequences have turned out to be appreciably more predictable; however, vector-induced leukemogenesis remains an unpredictable factor as it pertains to gene therapy, owing to its multifactorial nature. Nevertheless, there is a need to balance the glaring potential adverse effects against the clinical benefits for a particular patient, while putting the clinical complications associated with alternative treatments into perspective (e.g., allogeneic hematopoietic stem cell transplantation (HSCT) from a mismatched donor). The success along with the feasibility of gene therapy is undeniable, taking into account the benefits recorded with patients of different classes of diseases or disorders needing cure, including SCID-X1 and ADA-SCID, within the last two decades. PIDs (manifesting in the so-called bubble boys and girls) are rare without any doubt; nevertheless, they are life-threatening genetic diseases capable of severely compromising the integrity and functions of the immune system. This forces such individuals to live in a pathogen-free environment due to an inefficient immune system. PIDs targeted by gene therapy, which include ADA-SCID, SCID-X1, WAS, and CGD, occur in children with symptoms that include recurrent infections, system failure, and often death, most likely soon after birth. Bone marrow transplantation (using HSC) from human leukocyte antigen (HLA)-matched donors (<20% availability) has been a good alternative; however, the risks and complications associated with such strategies typically outweigh the benefits [13,14,15]. The alternative strategies that address these setbacks/limitations of scarce HLA-matched donors are found in the therapeutic potentials of autologous HSCs. As a result, this helps to avoid complications associated with immune suppression (IS) as well as the hurdle of graft against host infections [16,17,18,19].

Major setbacks and safety concerns are involved in the use of the existing murine γ-retroviral vectors designed for ex vivo gene transfer, leading to the onset of leukemia in a good number of patients who received treatment [20,21,22]. However, SCID-X1, which actually involves immunodeficiency disorders with T cells absent, natural killer (NK) cell deficiency, impaired function of B cells, and γ-chain (γc)-dependent cytokines, can be corrected by the reconstitution of immune competence in patients (for instance, via the application of SIN viral vectors). One of the core advantages of SIN is that they are devoid of LTR promoter/enhancer function, thereby reducing the risk of clonal dominance as well as insertion-related mutagenesis [23,24,25]. A similar application of the γ-retroviral vectors (murine) is present in ADA-SCID, a fatal PID with impaired T-, B-, and NK-cell growth, characterized by mutations in the gene coding for ADA (responsible for detoxifying the body of toxic purine metabolites), thus exposing patients to severe infections [9,16,17,18,26].

A number of ongoing clinical trials (#NCT01175239 (London), #NCT01410019 (Paris), #NCT01129544 (United States), #NCT01852071, #NCT01380990) are aimed at infusing interleukin-2 receptor γ-chain gene (IL2RG) to autologous HSCs in order to be transduced into SCID-X1 recipients to restore T-cell efficiency [26,27]. The same approach applies to WAS (#NCT01515462). There are also other areas where SIN-LVs are beneficially used. Autologous CD34+ HSCs derived from bone marrow, transduced ex vivo by means of LV-w1.6W vectors that convey a corrected copy of WASp gene, is one of the specific applications of LVs to WAS therapy. Compared with the γ-retroviral strategy for gene therapy in WAS, no clonal dominance or insertional mutagenesis was observed, and there is no need for a germ-free environment [28,29,30]. In addition to the gene correction in multiple cell lineages as well as the noticeably faster immunological reconstitution, the absence of detectable vector integration-associated adverse reactions is observed up to 3 years after close monitoring [31,32,33]. Approaches, such as employing a myeloid-specific promoter to avoid insertion-related mutation complications, are aimed at achieving better gene-corrected cell reconstitution and are also currently a critical area of focus. In these trials, however, some conditions could not be monitored effectively due to some other influencing conditions such as the metabolic effects of enzyme replacement therapy [9,16,34,35]. Low engraftment of gene-corrected cells in attempts to manage CGD (an uncommon genetic disorder because of a mutation in gp91phox subunit of nicotinamide adenine dinucleotide phosphate oxidase [NADPH]) has proven relatively unsuccessful due to the transient clinical benefits that it provides, as evidenced in the recurrence of the disease. The disease results in the failure of phagocytic cells (macrophages and neutrophils) to generate reactive oxygen species, hampering the efficient clearance of fungal and bacterial infections [36]. The use of γ-retroviral vectors for this disease also results in clonal dominance of gene-corrected cells capable of producing monosomy and myelodysplastic syndrome [27,36,37]. Most importantly, now is the time to put a spotlight on the most recent developments in this field as well as putting forth a road map for the future breakthroughs.

Vaccines are most commonly made up from the attenuated pathogens in forms administrable into a host’s system. However, some vaccines fail, and two main reasons could account for why vaccines fail after their preliminary development. The first is failure of the immune response, and the second is pathogenicity. The potency of a vaccine heavily relies on its ability to elicit an immune response just by recognition, while remaining nonpathogenic. However, the main cause of why vaccines fail, is that the vaccine candidate may not induce an immune response, as we lack full and comprehensive knowledge of the complexities of the host immune system. Second cause is the ability of the vaccine candidate to illicit an immune response by actually becoming pathogenic. In most PID cases, administration of subcutaneous or intravenous immunoglobulin is the primary choice of treatment. The main challenges with the use of vaccines in PIDs are the impairment of immune response, and impediment the immune system’s antigen stimulation, which results in very little or no protection caused by the immunodeficiency in some PID cases. In addition, adverse effects often result from vaccines as well as possible emergence of pathogenesis from vaccine strains. For example, in patients with phagocytic cell defects or defective T cells or NK cells (leukocyte adhesion deficiency), the immune response could be altered as a result of the live viral as well as live attenuated bacterial vaccines. This can create severe disorders linked to the vaccine strains, with the Chediak–Higashi syndrome and CGD being a common example.

3 CRISPR-Cas9 and the future of gene therapy

The CRISPR-Cas9 genome editing tool has recently become a common preference for gene therapy studies in basic research as well as in clinical trials. Though discussions are still ongoing concerning ethics in human applications, the CRISPR-Cas9 technology is undoubtedly a powerful tool for gene therapy when fully refined. CRISPR-Cas9 is an efficient site-directed genome editing tool mainly composed of Cas9 RNA-guided nuclease and CRISPR RNA (crRNA), which directs the Cas9 enzyme to the sequence of interest in the genome. This specificity is determined by a 20-nucleotide sequence complementary to the crRNA, following a protospacer adjacent motif sequence [84,85]. The CRISPR system uses the host cell’s innate error-prone nonhomologous end joining to achieve the aim. Many concerns regarding off-target and associated complications are still under review by experts to ascertain the safety of this genetic tool before it can be implemented in human trials. Genetic engineering and biotechnology companies are keeping a close look at the CRISPR-Cas9 tool as the technology has been used very recently in basic science research, and clinical trials are commencing in pharmaceutical research to use the CRISPR-Cas9 to treat patients with β-thalassemia [86].

Although CRISPR-Cas9 has proven to be an enormous scientific breakthrough, it has many limitations that have made it a frontline topic of discussion in research and medical applications, with legal implications. In 2015, an international summit on human gene editing supported the establishment of an international committee to assess the implications of CRISPR-Cas9. The Committee on Ethics, Law and Society of the Human Genome Organization on CRISPR-Cas9 produced some vital ethical points to consider in the application of CRISPR-Cas9, most particularly in its application to human biology and medicine [87]. On the bright side, CRISPR targets almost any nucleotide sequence with a short length of specific genes; however, it does not faithfully insert new target DNA sequences. Off-targets often occur, creating deleterious effects that are sometimes irreversible. This error leads to serious mutations such as in Friedreich ataxia, fragile X syndrome, and Huntington disease. As effective as it is, the CRISPR system can fail in identifying the right target, including introns, and as a result cause further damage. Challenges and ethical considerations about the use of CRISPR technology are not just peculiar to CRISPR but common to all gene editing tools previously employed. Experiences and challenges with gene editing raise discussion on delaying the application of the CRISPR-Cas9 in medical applications until well proven and tested using highly supported basic science [88,89,90,91]. For instance, an example is the emergence of childhood leukemia arising when the viral vector activated a latent human oncogene, as Double Strand Breaks (DSBs) generated by CRISPR-Cas9 often induces apoptosis. Some of the components of the CRISPR-Cas9 system, such as the cas9 protein, are of microbial origin and may trigger immune responses in the human system, which may be undetected for a longtime. The CRISPR-Cas9 system has been around only for a short time and requires more time to investigate all the implications associated with its use in genetic engineering.

4 Conclusions

In recent times, the field of gene therapy has recorded clinical achievements in various disease types by employing different methodologies and improved vectors. Recently, numerous gene therapy trials have validated the clinical benefits of the application of molecular biology and biotechnology tools in treating diseases or disorders that have proven elusive to cure using conventional pharmacotherapy. Such areas of application include hereditary immune system disorders, congenital eye diseases, hemophilia B, lipoprotein lipase deficiency, and adoptive transfer of genetically engineered T cells for cancer. Although gene therapy possesses obvious scientific, ethical, and technological challenges, many efforts have been made to facilitate the effectual translation of gene therapy into clinical practice. Both recent and noteworthy is the application of gene therapy to PIDs, hemoglobin, hemophilia B, ocular, and neurodegenerative disorders. Most recently, but still under strict refining, is the CRISPR-cas9 gene-editing tool; although it currently has some setbacks, it is promising at providing solutions to many genetic diseases. The application of gene therapy to these areas has pushed medical research and other applications toward becoming the remedy we have sought concerning such disease areas.