Haemophilia And Gene Therapy

Rationale

Gregory in the Daily Mirror claimed that ‘All inherited diseases could be cured within 20 years thanks to gene editing breakthrough’ (Gregory, 2017). After initial research, a draft question ‘Can Hemophilia A (HA) be cured with Gene Therapy?’ is posed.

“Gene editing” biotechnology is advancing rapidly, with potential cures already being developed. However, 20 years may be inadequate to develop cures for “all inherited diseases”. Hence, this report suggests a 50 year period. Also, the claim of curing “all inherited diseases” may be too broad, therefore this report focuses on Haemophilia.HA is a hereditary disorder causing impaired blood clotting. A mutation to the F8 gene, affects liver production of FVIII (Genetics Home Reference, 2019). Gene Therapy promises to cure HA, directly targeting the liver and F8 gene. With their ability to ‘infect’ cells, viruses are used to carry modified genetic material. They are genetically modified to prevent them causing illness (Genetics Home Reference, 2019).

The research question was further developed to specifically consider males (Centers for Disease Control and Prevention, 2019), method of cure and time. Hence, this essay addresses the question: ‘To what extent will Haemophilia A in males be able to be cured through Viral Gene Therapy and CRISPR-Cas9 in the next 50 years?’

Further Background ResearchHaemophiliaMild and Moderate HA is generally caused by point mutations and deletions to the F8 Gene, found at Xq28, or on base pairs 154,835,788 to 155,022,723 (X chromosome) (Bowen, 2002). Severe HA is generally caused by an inversion or insertion of genetic code, introns 1 and 22 (Tantawy, 2010). This mutation changes FVIII production, an important protein for clotting, resulting in increased bleeding (Genetics Home Reference, 2010).

As HA involves the X chromosome, females carriers rarely show symptoms, with their second X chromosome overriding the defect. Having a single X chromosome, males are more commonly affected (Centers for Disease Control and Prevention, 2018). For information on HA inheritance.

Gene Therapy

Viral Gene therapy corrects the F8 gene, creating more FVIII protein. A vector is injected directly into tissue where it penetrates cells. Alternatively, it is injected into patient’s cells externally and then returned to the body. The new gene then creates the required protein (Genetics Home Reference, 2019).

CRISPR-Cas9 therapy uses Cas9 (DNA-cutting enzyme), to edit specific genes. Target DNA is identified by the corresponding sequence on the guide RNA (transported with Cas9). This allows edited genes to be inserted into the genome (Ball, 2016), potentially curing diseases caused by gene mutations.

In this study, researchers injected a single dose of AAV5-hFVIII-SQ into nine HA men and monitored their status for 52 weeks on a four-weekly cycle. FVIII levels rose slowly initially. By week 16, most subjects reached therapeutic range. By week 20, levels continued to rise and remained within a mid-therapeutic range for the remaining trial period.

Additionally, specific FVIII information was collected with general patient data. This was used to monitor patient progress and further understand what variables may affect treatment success. See the table above.

Study 1, concluded that this therapy could sustain normal FVIII activity – six participants achieved a normal level (> 50 IU per decilitre), which was maintained for a year. Subsequently significantly less FVIII was required to further sustain their levels. This proves that a cure for Haemophilia is possible.

Study 2: Restoration of FVIII Expression by Targeted Gene InsertionReference for Section - (Sung et al., 2019) Study 2 involves a trial where iPSCs, TALEN and CRISPR-Cas9 were used to attempt to cure HA. By taking the mutated F8 DNA and inserting a corrected DNA sequence using iPSC’s to a specific location, a “normal” DNA sequence was created allowing normal FVIII function and clotting.

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Data is limited, involving a single patient with a specific gene mutation present in only some patients. With many different mutations causing HA, a new method would be required for each mutation.

Despite its limitations, results showed a successful outcome using iPSC’s for targeted gene insertion. Although this experiment targeted a specific mutation, the results could be extrapolated for other mutations, and diseases. It also showed that endothelial progenitor cells could express FVIII, mRNA and active FVIII protein. Thus, the use of a transgene could mean universal gene correction. Therefore, this report concludes that a cure for HA is likely in the near future.

Study 3: CRISPR to Correct Clotting in New-born and Adult MiceIn Study 3 mice with Haemophilia B were treated with an adeno-associated virus (AAV) carrying saCas9 and an RNA sequence to target the “5-prime end of exon 2 [mouse] of the FIX gene”. This CRISPR-mediated method repaired breaks on both strands of DNA and implanted human cDNA into the mouse FIX gene. After injection, the mice showed stable and normal FIX activity for over four months.

This study proves that CRISPR-Cas9 gene targeting could effectively treat patients with a specific disease like HA. It shows that a multi-vector approach is effective, achieving sustained normal liver function. See Trends.

Trends, Patterns and Relationships Across Studies

These studies detail different approaches to treating Haemophilia, providing evidence that gene editing has the potential for curing/managing HA. Both study 1 and 3 reported a delayed therapeutic response. However stable therapeutic levels were achieved for an extended time, achieving HA control.

The second study indicates that specific gene modification with iPSCs provides a potential cure. The specific gene modification for each individual patient is a complex procedure with a longer term positive outcome.

EvaluationStudy 1 provides long-term treatment, providing improved quality of life while patients await cure. This study had multiple subjects (9 men), with no noticeable issues, as evidenced by the information in ‘Study Design and Assessments’ (Rangarajan et al., 2017).

Study 2 suggests that Gene Therapy for disorders such as HA is likely in the near future. It’s main limitation was the lack of replication within the study (sole individual). Consequently, the success of this technique for other mutations is less predictable. This method could result in “low expression of FVIII” and “multiple FVIII transgene insertions in one cell.” (Sung et al., 2019). This could occur if a transgene overrides an important section of genetic code or introduces duplicate sections of code.

Study 3 suggests that future Gene Therapy for HA is likely. Mice achieved sustained normal FIX levels, suggesting the potential to cure HA and similar genetic disorders. A major limitation was that there was little data reported in the scientific article. With minimal evidence, results were unverifiable. There are no other errors of significance (Penn Medicine News, 2019).

These recent studies are from credible authors and organisations such as the New England Journal of Medicine and Penn University, and are somewhat recent tests, ensuring these results are accurate. There were limited reliable sources for HA specifically. A study on HB was included for this reason and to demonstrate a wider range of success in curing Haemophilia.

Conclusion

It can therefore be concluded that both managing and curing HA is possible through viral methods of Gene Therapy. Management is likely to be simpler to achieve than a cure. However, a cure will likely become achievable with scientific advancement. Therefore, my research question and Gregory’s claim that ‘All inherited diseases could be cured within 20 years thanks to gene editing’ are supported through evidence in this report.

References

1. https://www.cdc.gov/ncbddd/hemophilia/facts.htmlCenters for Disease Control and Prevention. (2018). What is Hemophilia? | CDC. [online] Available at: https://www.cdc.gov/ncbddd/hemophilia/facts.html [Accessed 21 Oct. 2019].
2. Gregory, A. (2017). All inherited diseases could be cured within 20 years due to gene breakthrough. [online] Daily Mirror. Available at: https://www.mirror.co.uk/science/inherited-diseases-could-cured-within-9832107 [Accessed 17 Oct. 2019].
3. Centers for Disease Control and Prevention. (2019). Data & Statistics - Hemophilia. [online] Available at: https://www.cdc.gov/ncbddd/hemophilia/data.html [Accessed 21 Oct. 2019].
4. Genetics Home Reference. (2019). What is gene therapy?. [online] Available at: https://ghr.nlm.nih.gov/primer/therapy/genetherapy [Accessed 21 Oct. 2019].
5. Genetics Home Reference. (2019). How does gene therapy work?. [online] Available at: https://ghr.nlm.nih.gov/primer/therapy/procedures [Accessed 21 Oct. 2019].
6. Robertson, S. (2018). Hemophilia Genetics. [online] News Medical. Available at: https://www.news-medical.net/health/Hemophilia-Genetics.aspx [Accessed 21 Oct. 2019].
7. Genetics Home Reference. (2010). F8 gene. [online] Available at: https://ghr.nlm.nih.gov/gene/F8 [Accessed 15 Oct. 2019].
8. Tantawy, A. (2010). Molecular genetics of hemophilia A: Clinical perspectives. Egyptian Journal of Medical Human Genetics, [online] 11(2), p.106. Available at: https://www.sciencedirect.com/science/article/pii/S1110863010000108#! [Accessed 4 Nov. 2019].
9. Bowen, D. (2002). Haemophilia A and haemophilia B: molecular insights. Molecular Pathology, [online] 55(1), pp.1-18. Available at: https://mp.bmj.com/content/55/1/1 [Accessed 4 Nov. 2019].
10. Ball, P. (2016). CRISPR: Implications for materials science. MRS Bulletin, [online] 41(11), pp.832-835. Available at: https://www.cambridge.org/core/journals/mrs-bulletin/news/crispr-implications-for-materials-science [Accessed 5 Nov. 2019].
11. StudiesRangarajan, S., Walsh, L., Lester, W., Perry, D., Madan, B., Laffan, M., Yu, H., Vettermann, C., Pierce, G., Wong, W. and Pasi, K. (2017). AAV5–Factor VIII Gene Transfer in Severe Hemophilia A. New England Journal of Medicine, [online] 377(26), pp.2519-2530. Available at: https://www.nejm.org/doi/full/10.1056/NEJMoa1708483 [Accessed 15 Oct. 2019].
12. Penn Medicine News. (2019). Penn Scientists Use CRISPR for First Time to Correct Clotting in Newborn and Adult Mice – PR News. [online] Available at: https://www.pennmedicine.org/news/news-releases/2016/november/scientists-use-crispr-for-first-time-to-correct-clotting-in-newborn-and-adult-mice [Accessed 16 Oct. 2019].
13. Sung, J., Park, C., Leem, J., Cho, M. and Kim, D. (2019). Restoration of FVIII expression by targeted gene insertion in the FVIII locus in hemophilia A patient-derived iPSCs. Experimental & Molecular Medicine, [online] 51(4). Available at: https://www.nature.com/articles/s12276-019-0243-1 [Accessed 16 Oct. 2019].