GEN124 Genetics

Introduction

Sickle cell anaemia is a common and chronic genetic disease with substantial mortality rate and morbidity. It is caused by the mutation in β-globin alleles. This can happen in either or in combination of homozygous and heterozygous. Foetal haemoglobin is passed down by hereditary persistence which caused the genetic mutation in the gene cluster of β-globin (Dever et al. 2016). Sick cell decreases the deformability of red blood cells and forms shape of a sickle which triggers an extremely painful vaso-occlusive crisis leads to severe pain which in turn leads to multiple organ damage. Sickle cell anaemia have affected around 90,000 individual at United States of America and annually 275,000 infants or more born with sickle cell anaemia (Ribeil et al. 2017). This prompts numerous investigation regarding the treatment for sickle cell anaemia in both academic, government and private sector. Unfortunately, there is no cure for sickle cell anaemia at present. Only short term treatment that are available for the sickle cell anaemia are stem cell or bone marrow transplantation. This is an expensive process and only available to the few patients with suitable and compatible donor. However, less than 18 per cent patients amongst the affected are beneficial to this treatment (Ribeil et al. 2017).

With the development of whole human genome sequencing, bioinformatics and technological advancement of new and affordable genome sequencing method, genome editing have emerged as an alternative method to find cure for sickle cell anaemia. Recently, researchers are still unable to find the molecular mechanisms which affects or influences a phenotypes. Genome editing can tackle this problem. Genome editing is a way to supress or shut down a function of a specific gene by editing a specific DNA sequence. It is concerns the editing of programming of highly specific nucleases which incites the site specific changes in the cellular organism’s DNA. Genome editing is obtained through site specific DNA binding domain and non- specific cleavage domain of DNA. There are various genome editing process developed now a days. These include Zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system (Gaj, Gersbach and Barbas III 2013).

At present, CRISPR/Cas9 have been emerged as an alternative methods to the Zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). CRISPR/Cas 9 depends on the function of crRNA and tracrRNA for the invasion and site specific silencing of foreign DNA. At present, three types of CRISPR/Cas9 genome editing system is available (Gaj, Gersbach and Barbas III 2013).

Therefore, in this review article gene therapy and CRISPR/Cas9 genome editing system will be discussed thoroughly as alternative treatments for sickle cell anaemia. To achieve that different application methods, theories and techniques will be reviewed from the published peer reviewed articles.

Discussion

In this section, thorough description and analysis of gene therapy method and CRISPR/Cas9 genome editing techniques for sickle cell anaemia will be discussed.

Gene therapy

Academic researchers have long been praised gene therapy as a potential and viable treatment plan for sickle cell anaemia. One of the methods for gene therapy is the addition of gene (Loucari et al. 2018). For the successful achievement of this method two key principles should be addressed. These two methods are: i) transfer of gene in an efficient and safe manner and editing of the long term repopulation of haematopoietic stem cells and ii) stable, highly effective and properly regulated gene expression. Sickle cell anaemia occurs because of the mutation in β-globin gene cluster, hence, modification performed in β-globin, γ-globin and other antisickling vector will be discussed below (Hoban et al. 2016).

β-globin expression in vectors

Principle findings that allowed this β-globin expression in vivo is the characterization and discovery of β-globin locus control region (LCR). This is a 40 to 60 kb long regulatory element in β-globin gene. Mapping of the activities and positions of the hypersensitive sites present in the β-globin gene enables the expression of the high level erythroid (Cavazzana,  Antoniani and Miccio 2017). One of the challenges for the β-globin expression in vectors is that the recognition of the silencing and variegation expression. Insulators presents in the hyper sensitive sites shields and blocks the element alpha and beta by decreasing the trans- activation. Balancing this act is the ongoing issue of the researchers. Figure 1 depicted the various vectors (lentiviral here) carrying the β-globin gene of human which are used for correction of disease like sickle cell anaemia.

Figure 1: Lentiviral vectors which are used for correction of disease in human.

Source: (Hoban et al. 2016)

γ-globin and other antisickling

Apart from using β-globin vectors, other vectors like γ-globin or other hybrid vectors are being developed by the researchers. The rationale behind this development is the foetal haemoglobin like HbF are much more potent antisickling haemoglobin in comparison with the adult haemoglobin. It has been found that HbF level is higher in sickle cell anaemia patients. A study have shown that beta and lamda globin expressing lentiviral vectors have corrected phenotypes of red blood cell of sickle cell anaemia which suggested that this vector is viable for clinical development which compares the betaAS3-FB vector and V5m3-400 vector (Urbianti et al. 2015). This kind of direct comparison is needed for the proper development of the plan.

The current challenges for the gene therapy is that numerous researchers have been working in this field and all of them have different approach to this solution with regards to gene, vector, experimental model, regulatory elements, experimental model, insulators and many other parameters. A unified and collaborative method or standardized procedure is needed for the successful implementation of gene therapy as a potential cure for sickle cell anaemia.

Recent sickle cell anaemia gene therapy clinical trials

Three research groups have been registered in the clinicaltrials.gov for the gene therapy for sickle cell anaemia. These three groups are Bluebird Bio, University of California, LA and Clinical Children’s Hospital Medical Centre. Bluebird Bio which is a biotechnological company is the first company to treat a sickle cell anaemia disease patient with gene therapy (Archer, Galacteros and Brugnara 2015).  They used the vector LentiGlobin BB305 which expresses the T87Q antisickling β–globin gene (Negre 2015). Their subject, a 13 year old individual have not reported any side effects after 4 and half months of treatment. Clinical trials performed by other two organisation are open currently and they have reported any of their findings yet. University of California used 6 adult subjects whereas Clinical Children’s Hospital Medical Centre institute used 10 adult subjects with age 35 years old.

Genome editing by CRISPR/Cas9 techniques

Currently, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become widely used, easily applicable and developed vastly over the past few years. Presently, It is the most efficient and simple tool for genome editing. CRISPR/Cas9 mediated genome modification is needed for successful application of genome editing. In this section, CRISPR/Cas9 mediated genome modification, application and challenges of CRISPR/Cas9 genome editing will be discussed.

CRISPR/Cas9 mediated genome modification

CRISPR/Cas9 present in archaea and bacteria act as an acquired immune system for phages and viruses. Out of all the genome sequence of bacteria, CRISPR/Cas9 present in the nearly 40 per cent of them and almost 90 per cent in archaea. In this system, foreign DNA is processed by the Cas nuclease system into fragments of DNA which is then incorporated into the locus of CRISPR region of the host genomes. This transcriptional templates is responsible for the production of the crDNA which is then guides the Cas to cleave the target DNA sequence of invading phages and viruses. Generally, CRISPR/Cas9 system broadly divided into three types which are known as Type I, Type II, and Type III (Zhang, Wen and Guo 2014).

Genome editing by CRISPR/Cas9 depends on the double strand break (DBS) generation and following repair process of cellular DNA. crRNA present in CRISPR/Cas9 system is combined with tracrRNA to form tracrRNA:crRNA complex which guides the Cas9 complex to its target sites. Then, at the target site, CRISPR/Cas9 mediated sequence cleaves the target DNA sequence. Over the year, researchers have engineered and developed various 20/ 24 nt sequences specific to gRNA and 2 to 4 nt PAM sequence at the target sites. Hence, theoretically, CRISPR/Cas9 system can uniquely identify 22 to 29 nt long DNA sequence which are unique to most genomes. Although, researchers have reported that CRISPR/Cas9 system has high tolerance against base pair to its complimentary DNA sequence. To cite one example, Streptococcus pyogenes’s CRISPR/Cas9 system is tolerant up to 6 base pair mismatch (Zhang, Wen and Guo 2014). In the Figure 2, a schematic diagram of CRISPR/Cas9 mediated DNA cleavage has been depicted.

Figure 2: Schematic diagram of CRISPR/Cas9 system mediated DNA cleavage.

Source: (Zhang, Wen and Guo 2014)

This figure depicts how a mature crRNA directs Cas9 to its target sites of the foreign DNA which appeared by the invasion of phage DNA and how the total cleaving process occurs.

Application CRISPR/Cas9 in genome editing

CRISPR/Cas9 system is a multiplex-able and robust editing tool which helps researchers manipulates precisely a specific genomic sequence. This assistances the elucidation of target gene for disease like sickle cell anaemia. Research evidence have already reported many successful mutation caused by CRISPR/Cas9 in various mammalian genome and plants. For instance, Ye et al. (2016) have used genome editing by CRISPR/Cas9 system to treat sickle cell anaemia. They have used a 13kb long β-globin locus to mimic the mutation of hereditary persistence of foetal haemoglobin (HPFH). Their target detection efficiency reached up to 31 per cent. In their study, the authors have suggested that their approach can be used to safe transplantation for the sickle cell anaemia patients. On the other hand, Dever et al. (2018) have used CRISPR/Cas9 β-globin gene targeting using human haematopoietic stem cells. In their study, the authors have claimed that their methodology can be used not only for the treatment for haemoglobopathies but also for other haematological diseases. Genome editing by CRISPR/Cas9 system can be done by gene disruption, gene addition and gen correction. Table 1 presents few examples of the CRISPR/Cas9 mediated genome editing have been performed in human till today.

Table 1: Examples of CRISPR/Cas9 mediated genome editing in human cell

Challenges of CRISPR/Cas9 genome editing

Although, CRISPR/Cas9 has great potential, it is not without few issues which need to be addressed which are PAM dependence, off target mutations, delivery methods and gRNA production.

Pam dependence: This is one of the significant issues that CRISPR/Cas9 genome editing system has as specificity of the CRISPR/Cas9 depends on the 2 to 5 nt long PAM sequence. Additionally, PAM sequence varies among the different Cas9 protein orthologs. Interestingly, this also increases the specificity of the CRISPR/Cas9 system. From the evidence reported by Hsu et al. (2013) NAG PAM has only about 20 per cent efficacy of NGG PAM for guiding DNA cleavage.

Off target mutation: This is the most primary and significant concern for genome editing by CRISPR/Cas9 system. It has comparatively high risk of off target mutation in human cell in comparison with other genome editing tools like ZFNs and TALENs. This is very vital as off target mutation can lead to transformation or cell death. To counter this, Xiao et al. (2014
) have developed a searching tool named CasOT which finds the genome sequence in the genome which can be potential off target sites.

gRNA production: Production of gRNA is another major issues for the CRISPR/Cas9 genome editing as it requires extensive modification and processing of post  transcriptional mRNA which transcribed by RNA polymerase II. However, RNA polymerase III is being used in vivo for the production of gRNA. Lack of commercially available RNA polymerase III also hinders the production gRNA.

Delivery methods: Concerns also remains regarding the delivery methods of CRISPR/Cas9 system in the organism as it depends on the target component such as tissues and target cells. Research gap remains in this particular area and much research focussed in this region for the development efficient delivery methods for CRISPR/Cas9 based genome editing.

Conclusion

Therefore, from the above discussion, it can be said that gene therapy and CRISPR/Cas9 mediated genome editing has emerged as potential treatment plan for the sickle cell anaemia as both techniques have developed technologically and overcame some early obstacles. On one hand, Gene therapy using transduction haematopoietic stem cells with lentiviral vectors for the treatment of sickle cell anaemia as it can provide stout transduction. Clinical trials for these methods are already in underway. On the other hand, genome editing by CRISPR/Cas9 system has the potential for facile and reliable editing tool as it is efficient, simple and requires low cost assembly of nucleases. This adaptability and simplicity can lead to the pathway for the successful treatment plan for sickle cell anaemia. However, both the techniques have their limitation which requires further investigation. Hence, to summarize, it can be concluded that both the gene therapy and CRISPR/Cas9 genome editing tool are viable and has potential to be the long term treatment techniques for sickle cell anaemia but both the techniques needed further investigation and development to iron out the limitation of this techniques which are hindering their advancement.

References

Archer, N., Galacteros, F. and Brugnara, C., 2015. 2015 Clinical trials update in sickle cell anemia. American journal of hematology, 90(10), pp.934-950.

Cavazzana, M., Antoniani, C. and Miccio, A., 2017. Gene therapy for β-hemoglobinopathies. Molecular Therapy, 25(5), pp.1142-1154.

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Gaj, T., Gersbach, C.A. and Barbas III, C.F., 2013. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in biotechnology, 31(7), pp.397-405.

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Negre, O., Bartholomae, C., Beuzard, Y., Cavazzana, M., Christiansen, L., Courne, C., Deichmann, A., Denaro, M., de Dreuzy, E., Finer, M. and Fronza, R., 2015. Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease. Current gene therapy, 15(1), pp.64-81.

Ribeil, J.A., Hacein-Bey-Abina, S., Payen, E., Magnani, A., Semeraro, M., Magrin, E., Caccavelli, L., Neven, B., Bourget, P., El Nemer, W. and Bartolucci, P., 2017. Gene therapy in a patient with sickle cell disease. New England Journal of Medicine, 376(9), pp.848-855.

Urbinati, F., Hargrove, P.W., Geiger, S., Romero, Z., Wherley, J., Kaufman, M.L., Hollis, R.P., Chambers, C.B., Persons, D.A., Kohn, D.B. and Wilber, A., 2015. Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells. Experimental hematology, 43(5), pp.346-351.

Xiao, A., Cheng, Z., Kong, L., Zhu, Z., Lin, S., Gao, G. and Zhang, B., 2014. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics, 30(8), pp.1180-1182.

Ye, L., Wang, J., Tan, Y., Beyer, A.I., Xie, F., Muench, M.O. and Kan, Y.W., 2016. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia. Proceedings of the National Academy of Sciences, 113(38), pp.10661-10665.

Zhang, F., Wen, Y. and Guo, X., 2014. CRISPR/Cas9 for genome editing: progress, implications and challenges. Human molecular genetics, 23(R1), pp.R40-R46.