Huntington Disease: Overview

Huntington disease (HD) also known as Huntington choera derives its eponymous title from Dr. George Huntington, the discoverer of the disease (Bates, Tabrizi and Jones 2014). It is an autosomal dominant disease cause by the defects in the short arm of chromosome 4 (4p16.3) [350 Kda]. It is however, effective in both heterozygote and homozygote however; homozygotes are more severely affected than heterozygote (Bates, Tabrizi and Jones 2014; Bates et al. 2015). The gene associated with the Huntington disease (HTT) contains tri-nucleotide repeat (TRE), CAG (cysteine-adenine-guanine) at the 5’ region of exon 1 and codes for the protein huntingtin (Bates, Tabrizi and Jones 2014). Alleles containing repeat lower than 25 is not associated with disease onset. Alleles containing 26 to 35 repeats, do not lead to disease manifestation but shows mitotic instability (Lee et al. 2012). However, allele more than 35 is associated with disease progression. Repeats more than 50 are associated with early onset, juvenile Huntington (Lee et al. 2012). CAG encodes for Glutamine so, overexpression of CAG repeat at the 5’end of the gene leads to the increase in the poly-glutamine tract in this ultimately lead to protein misfolding. This protein folding anomalies is expressed as slowly progressive movement disorder including hyperkinetic, involuntary movements, dystonia and chorea (Bates, Tabrizi and Jones 2014). The protein misfolding also confers toxic gain of function which eventually predisposes the protein towards the subsequent process of fragmentation resulting in the development of neuronal dysfunction along with insidious impairment of intellect along with psychiatric ultimately leads to death (Bates et al. 2015).

Following report provides a layout of systematic literature review that is based on the how RNA suppression works in animal models of Huntington’s disease and problems translating this work to humans. While doing the systematic review, the report will review two papers which can conducted their works based on the RNA model of disease treatment in relation to Huntington disease. The review will be based on the critical analysis of methodology employed for the study, difficulties in methodology and presence of bias if any and how it has affected the study. At the end, the report aims to provide recommendation based on the future prospects of the research work in Huntington disease treatment using advanced procedures of molecular biology and recombinant DNA technology.

Paper 1

Sustained Therapeutic Reversal of Huntington’s Disease by Transient Repression of Huntingtin Synthesis

According to Kordasiewicz et al. (2012), gene silencing technique via short hairpin RNA (shRNA) or micro-RNA is used as a potential therapeutic approach to supress the synthesis of faulty huntingtin gene. Further research by Kordasiewicz et al. 2012 demonstrated that transient infusion of antisense oligonucleotide (ASOs) via cerebrospinal fluid into symptomatic HD mouse model leads to RNase-H mediate degradation of huntingtin mRNA. This treatment delays the progression of the disease along with sustained reversal of the disease phenotype that stays longer in comparison to Huntington knockdown (Kordasiewicz et al. 2012).

Paper 1: Sustained Therapeutic Reversal of Huntington’s Disease by Transient Repression of Huntingtin Synthesis

Thus the main aim of study conducted by Kordasiewicz et al. (2012) is measure the extent of reduction in the synthesis of faulty huntingtin protein under the action of single stranded (SS) ASOs, infused into the central nervous system CNS in mouse model. The AOS infused is a 20-mer phosphorothioate modified oligonucleotide which is complementary with the mRNA of human huntingtin gene. In order to increase the stability of the ASOs inside the body, ethyl modifications on over 5 nucleotides from 3’ to 5’ end was done. The infusion was done continuously for 2 weeks in mutant model with 97 CAG repeats. The results showed that infusion of ASOs (50 microgram/day for 2 weeks) decreased the secretion of huntingtin mRNA in a dose dependent manner. The stability of the ASOs inside the mouse body was verified via gel electrophoresis. The reduction of huntingtin gene expression was observed for 12 consecutive weeks but rose to untreated level after 16 weeks after the termination of treatment (Kordasiewicz et al. 2012).

There was no significant methodological difficulty as the cellular uptake ASOs inside the mouse body was re-verified via selective antibody test which recognizes phosphorthioate backbone of ASO. Moreover, the mouse model that was used recapitulates human HD expression. The said treatment also helped in the improvement of the motor skills of the mice. In order to verify the longevity of the treatment, a second study was carried out over a second set of cohort fir 2 weeks. The same treatment was again repeated into more close primates of human, Rhesus monkeys and similar results were reciprocated (Kordasiewicz et al. 2012).

However, this paper has certain limitation in terms of results interpretation and possible hypothesis. This is because, the brain of Rhesus monkey though larger than brain of the mouse but is 1/15 of the brain of the human. Here the inhibition of the huntingtin gene takes place in a dose dependent manner. So the possible interpretation of the same results in human brain is a complex and proper standardization of dose and duration of infusion is subject to further analysis (McGonigle and Ruggeri 2014). Moreover, such AOs assay help in the reduction of both mutant and normal allele. Normal HTT gene is known to responsible for maintain ganglia anatomy and behavioural repertoire and thus can cast a threat towards human being (McBride et al. 2011).

Paper 2

Preclinical Safety of RNAi-Mediated HTT Suppression in the Rhesus Macaque as a Potential Therapy for Huntington ’s disease

Paper 2: Preclinical Safety of RNAi-Mediated HTT Suppression in the Rhesus Macaque as a Potential Therapy for Huntington’s Disease

The aim of the study conducted by McBride et al. (2011) is to investigate whether the partial reduction of HTT in a healthy non-human primate is safe. Previously genetically engineered exogenous miRNA helped in a robust decrease in the gene expression via RNA interference approach (RNAi). However, this strategy helped in the global inactivation of both the mutant and normal HTT gene. The normal HTT gene plays an important functional role in adult brain. Thus non-allele-specific reduction of HTT expression can considered to be safe. Complementary sequence for HTT gene was cloned into the artificially designed miRNA and was then subsequently injected inside the model organism via adeno-associated viral vectors. This recorded 45% reduction in the HTT expression of the rhesus in the caudal and mid putamen. On the other hand, this does not included the any case of neuronal degeneration, motor deficits and astrogliosis.  

The main limitation of the study include, it taken the approach of viral vector mediated delivery of the miRNA. Adeno-associated viral (AAV) vector that is used for the study is associated with certain limitations in gene delivery (Vannucci et al. 2013). For example adenovirus contains rep gene that is responsible for viral replication along with structural gene expression and chromosomal integration. The unmodified AAV integrates at specific site into the chromosomal DNA and this specificity of attributed under the presence of rep genes. However, rep gene is deleted in the construct and hence the integration of the target gene takes place randomly (Vannucci et al. 2013). Other disadvantages include difficulty in achieving in higher titters (which crucial in case of gene targeting in human) and requires co-infection via helper virus like adenovirus or herpes simplex virus (Vannucci et al. 2013). Lentiviral vector, another mode of vector used for the study also has certain disadvantages like threats towards positional mutagenesis, transient expression of transgene with integration-defective vector and presence of regulatory protein in packaging construct that may cast immunogenic threats (Vannucci et al. 2013).

Conclusion

HD is incurable. Numerous therapies have shown promise in mouse and rodent models of the disease but the majority of them failed to cast a major impact on the prevention of disease or extension of the projected life span when tallied in the clinical trial. As a result the present strategies od treatment are mostly directed towards the palliative care to improve the symptoms of the disease and improve the end-stage quality of life. This is done via reducing the expression of the harmful HTT gene itself that is found to have greater impact in clinical grounds in comparison to the strategies which are aimed towards targeting the downstream consequences of mHTT. Thus RNA mediated gene targeting and inactivation is now considered to be one of the most effective medium towards the treatment of the HD. However, in spite of getting promising results in the non-human primates, the similar reciprocation of similar results in human might be difficult to attain due to threats coming from the viral vector or the mode of delivery of the ASOs.

At present anaplerotic therapy is on trial and is projected as the potential treatment for the HD disease and other disease associated with the elongation of the polyglutamine tract. This therapy deals with the administration of an effective amount of propionyl CoA precursor to an individual suffering from HD. Propionyl Coa once injected inside the body, gets converted into ketone bodies under the action of the liver enzymes. These C-5 ketone bodies promotes normalization of glutamine under the action of GABA under the brain pathology of HD. This normalization of glutamine helps in controlling the adverse effect of HD. The precursors of propionyl-CoA an easily be administered orally, intraperitoneally or parentally. However, oral ingestion of propionyl-CoA is mostly preferred (Durr et al. 2018).

References

Bates, G., Tabrizi, S. and Jones, L. eds., 2014. Huntington’s disease (No. 64). Oxford University Press (UK).

Bates, G.P., Dorsey, R., Gusella, J.F., Hayden, M.R., Kay, C., Leavitt, B.R., Nance, M., Ross, C.A., Scahill, R.I., Wetzel, R. and Wild, E.J., 2015. Huntington disease. Nature reviews Disease primers, 1, p.15005.

Durr, A. and Mochel, F., Institut National de la Sante et de la Recherche Medicale (INSERM), 2018. Anaplerotic therapy of huntington disease and other polyglutamine diseases. U.S. Patent Application 15/640,018.

Kordasiewicz, H.B., Stanek, L.M., Wancewicz, E.V., Mazur, C., McAlonis, M.M., Pytel, K.A., Artates, J.W., Weiss, A., Cheng, S.H., Shihabuddin, L.S. and Hung, G., 2012. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron, 74(6), pp.1031-1044.

Lee, J.M., Ramos, E.M., Lee, J.H., Gillis, T., Mysore, J.S., Hayden, M.R., Warby, S.C., Morrison, P., Nance, M., Ross, C.A. and Margolis, R.L., 2012. CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion. Neurology, 78(10), pp.690-695.

McBride, J.L., Pitzer, M.R., Boudreau, R.L., Dufour, B., Hobbs, T., Ojeda, S.R. and Davidson, B.L., 2011. Preclinical safety of RNAi-mediated HTT suppression in the rhesus macaque as a potential therapy for Huntington’s disease. Molecular Therapy, 19(12), pp.2152-2162.

McGonigle, P. and Ruggeri, B., 2014. Animal models of human disease: challenges in enabling translation. Biochemical pharmacology, 87(1), pp.162-171.

Vannucci, L., Lai, M., Chiuppesi, F., Ceccherini-Nelli, L. and Pistello, M., 2013. Viral vectors: a look back and ahead on gene transfer technology. New Microbiol, 36(1), pp.1-22.