Haemophilia is a bleeding disorder that is caused by the absence of the gene encoding coagulation factor VIII or IX which is vital for the clotting in the blood, both proteins are produced in the liver by hepatocytes and endothelial cells (Nienhuis AW et al 207). The deficiency or absence of these factors can result in bleeding in the joints and soft tissue. Without treatment, it can cause chronic arthropathy and in severe cases death. In order to battle this disorder patients with VIII deficiency {Haemophilia A} are treated with replacement therapy products which are injected every 2-3 times per week, whereas IX {Haemophilia B} requires replacement therapy twice per week (Arruda et al 2020). This treatment is effective in the inhibition of spontaneous bleeding episodes but it is not the initial cure for this disorder as it is causes complications for the development of the inhibitors resulting in them being ineffective. Due to this treatment method being very expensive not all patients can access it all over the world, causing a reduced life expectancy for those who suffer from severe cases of haemophilia (Ponder KP et al 2008). However, a long-lasting treatment has been sought after through gene therapy using animal models.
Haemophilia is a perfect model disease for gene therapy trails due to its monogenic nature and calculable clinical endpoints such as the clotting factor and bleeding episode. In addition, it supports its ability to improve outcomes with the success of raising factor levels. The most recent clinical studies are based on the use of recombinant adeno-associated viral (rAAV) vectors which have shown success (Arruda et al 2020). To date treatment for haemophilia has mainly been focused on liver-directed transgene expression which are controlled by the liver-specific enhancers that restrict the expression of hepatocytes. Haemophilia rAAV vectors are produced either through the transfection of mammalian cells with naked plasmid DNA or through the introduction of baculovirus expression vectors into Spodoptera frugiperda(sf9) insect cells, trailed by cell lysis and purification via caesium chloride (Arruda et al 2020) (Colella P et al 2017). These classifications have eased the use of rAAV vectors for gene therapy in haemophilia (Grieger JC et al 2016).
Haemophilia B is the ideal target for gene therapy treatment, especially due to the fact that with little increment in the plasma factor IX levels over 1% of physiologic levels improves the extreme dying phenotype. When looking at vectors that are based on adeno-associated infections (non-pathogenic parvovirus) have appeared to be the most prominent for gene therapy in patients with haemophilia B, generally because the ability of these vectors to intervene in the long-term expression of factor IX after a single infusion. “Previously in early trails where gene therapy for haemophilia B utilized either intramuscular or liver targeted delivery AAV factor IX vectors based on AAV serotype 2″(Amit C Nathwani et al 2014). Results from this method showed that patients with severe haemophilia B they were not able to stabilize the expression of factor IX in the plasma. Moreover the toxicity levels of the liver were watched in patients within the liver targeted study, which could have been due to the activation of capsid-specific T cells which were infused with high vector dosage (Amit C Nathwani et al 2014).
Clinical case study
A case that was conducted on 10 patients that suffer from severe haemophilia B, the systematic implantation of AAV8 vector containing codon-optimizing factor IX transgene produced a long-term expression for factor IX at medicinal levels. A single intervention of the sample scAAV2/8-LP1-hFIXco brought about a growth in plasma factor IX activity from standard levels that extended from less than 1% of ordinary value to steady levels of 1-6% value in all patients (Amit C Nathwani et al 2014). Among them were patients who received low or normal doses of the vectors which resulted in a moderate increase of the IX factor. In contrast, the factor IX expression was observed in the patients who received a high dosage, the AAV8 mediated IX expression was stable over the periods of years as well as the transgene expression in both animal models and nonhuman primates. (Amit C Nathwani et al 2014)
Of the patients seven were receiving prophylaxis that had a concentration of factor IX prior to the gene transfer, four of the patients were able to stop the regular IX replacement therapy while the others increased the interval of prophylactic infusions. Although the use of factor IX concentration was reduced the yearly bleeding episodes were evidently lower after the gene transfer, most noticeable in patients with higher dosage whose IX expression was around 5% of normal value. This reduction also took place with higher physical activity from all patients including sports activities that were related to bleeding episodes. Patients have either no or exceptionally few bleeding episodes. (Amit C Nathwani et al 2014)
Table 1. characteristics of patients from baseline and after gene transfusion.
The table states that only patient 7 had immunity to hepatitis B after the vaccination and that patients 1,4 and 6 had a previous infection of hepatitis C. This table indicates that after the vector dosage was given to the patient’s none of them had any symptoms before and after administration especially those linked with a viral infection. (Amit C Nathwani et al 2014)
Figure 2 A&B
You can see here the effect of gene transfer on factor IX concentration and number of bleeding episodes before and after gene transfer and how It affects each dosage group. The annual bleeding episodes for patients with high dose decreased from a median of 16.5 episodes to 1.0 episodes. (Amit C Nathwani et al 2014)
AAV vectors
AAV vectors are obtained from wild type of AAV which are members of the parvovirus family, they are non-pathogenic, have replication deficiency and receive help from a virus so that they can replicate. AAV vectors deliver a transgene that is therapeutic to nondividing and diving cells (Perrin GQ et al 2019). The majority of these vector genomes do not mix with the host genome slowly resulting in progressive loss of transgene. AAV serotypes are separated into branches depending on their viral capsid homology, the viral capsid controls the tissue tropism and the intracellular particle trafficking, whereas engineered AAV capsid are generated by random mutagenesis and capsid gene shuffling. These have been created to help enhance human hepatocyte tropism evading neutralizing antibodies and elimination by cytotoxic T cells. AAV vectors that are liver-directed gene therapy in animal models induce immune tolerance to FVIII and FIX through factor specific regulatory T cells. New evidence states that AAV and LV (lentiviral) vector can eliminate established inhibitors through liver-directed gene therapy (Perrin GQ et al 2019).
Immunology of AAV gene therapy
Humans are all infected with wild-type AAV vectors which develop neutralizing antibodies (NABs) from childhood onwards. These often react with multiple serotypes that prevent gene transfer with AAV vectors. The NABs development on gene transfer also averts the patient’s chances of administrating the same AAV vectors. By activating the capsid-specific CD8+T cells it may cause the targeting of transduced hepatocytes and transient transaminitis and the loss in factors. To reduce the chances of this happening immune suppression protocols were developed based on drugs that are administered to increase transaminases in the blood (Amit C Nathwani et al 2011).
In comparison to T-cell assays by increasing the levels of the transaminases it allows for more rapid treatment. Vector dose and transgene activity levels play a significant role in whether the immune response is brought against AAV vectors and products of the transgene. Recent studies show that to date there hasn’t been any detection of B/T-cell response against FVIII OR FIX (Miesbach W et al 2018). Transgene expression in the hepatic environment can also lead to immune resilience to the protein antigen (Amit C Nathwani et al 2011).
The reasoning for gene therapy for haemophilia
Gene therapy duplicates the disease-causing gene which is either expressed as absent or non-functioning making it profoundly viable in treating haemophilia. Haemophilia is well suited for gene therapy as a treatment method due to the bleeding phenotype which is responsive to a number of factor levels. It can also help correct the clotting factor by gene delivery and hepatocytes as the clotting proteins are secreted into the blood circulation. The VII and IX factors can be synthesised in noneffective cells. Naturally, FVII and FIX are secreted by specialised endothelial cells which then generate a functional protein that reinstates hemostasis in the patients. Gene therapy can also be helpful to other countries by providing patients with a long-term treatment instead of single dosage treatment which are more expensive (Perrin GQ et al 2019).
Of the many ongoing Haemophilia B clinical trials, the first long term success for haemophilia gene therapy was sponsored by the university of college London and St. Jude children’s research hospital, for the patients they used scAAV2/8-LP1-hFIXco vector. The first patient was treated in 2009 making this case the longest follow-up on the stability and safety of FIX protein expression on AAV vector in the liver. The case also had many patients that had an early decline in FIX levels which were related to transient transaminitis which was related to the capsid T-cell response (Nathwani AC et al 1994-2004) (Miesbach W et al 2018).
Challenges of AAV- based gene therapy
The main safety factor related to clinical trials of gene therapy is the dosage given to patients and how it elevates liver transaminase after the infusion. In order to prevent this, most studies have used prophylaxis to aid protect the transduced hepatocytes. Another safety factor is the loss of transgene expression despite the intervention prior to dosage (Paul Batty et al 2019). There is also a risk of insertional mutagenesis after AAV- mediated gene transfer as the majority of vector genomes require episomal (Li H et al 2011).
Update on clinical gene therapy for haemophilia B
Given the recent update on gene therapy for haemophilia B, there are still disagreements regarding the benefit of the treatment and how the enhanced half-life factors have failed to show the benefits of half-life in FIX. In haemophilia B recent clinical trials have reported that there have been more stable therapeutic expressions for FIX. It is still unclear how AAV gene therapy will be in young kids that have less than 50-day exposure. Due to the risk of inhibitors and potential loss of the factor expression which would over time result in AAV-transduced hepatocytes during the growth of liver (Perrin GQ et al 2019).
An alternative for this could be LV which could offer a substitute vector platform to help treat children with haemophilia B within Vivo liver gene delivery of immune stealth (Perrin GQ et al 2019). It is still unclear whether the strong AAV mediated liver tolerance which was observed in animal models will work in children and adults with high-risk mutations for inhibitor. This proves that there is still a need for improvement in bypassing agent factors. (Perrin GQ et al 2019). Going forward with the research UCL has started a new phase ½ trial for haemophilia B by using undisclosed AAV-F9 vector (Nathwani AC et al 1994-2004). Gene editing approaches for haemophilia treatments are being investigated to correct the endogenous of F9 mutation (Park CY et al 2016).
Conclusion
Overall, with the clinical trials for the treatment of haemophilia B and the recent breakthroughs for haemophilia therapies, there are still debates questioning the benefits of gene therapy, also taking into consideration the approved enhanced half-life factor. This has proved to show that there is no record of success for improved half-life products in FVIII. However, there is still a great deal of interest regarding gene therapy for haemophilia using AAV vectors as a therapeutic method as there are several ongoing studies that are evaluating various vectors for the delivery of gene therapy.
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