Written by Dr. Ambika Tewari Edited by Dr. Hayley McLoughlin

Lentiviral expression of an shRNA against ataxin-3 was well-tolerated and produced no measurable adverse effects in wild-type mice.

Evaluating the safety profile is a necessary and crucial step in qualifying a therapy for use in patients. Gene therapy is an experimental technique that has demonstrated tremendous progress in the treatment or reversal of a disease, specifically monogenic disorders. Carefully investigating the safety and tolerance of gene therapy is important to gauge its suitability for clinical trials. Gene therapy tools can be used in different ways to achieve the same therapeutic effect: the faulty gene can be replaced with a healthy copy, the mutated gene can be repaired, or the mutant copy of the gene can be silenced. You can learn more about gene therapy in this pat SCAsource Snapshot.

Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) causes progressive loss of neurons in the spinal cord, and several regions of the brain. This includes the cerebellum, brainstem, striatum and substantia nigra. These neurons have crucial functions. Without these neurons, patients experience motor incoordination, loss of balance, and in severe cases, premature death. While great progress continues to be made in understanding how a mutation in a single gene, Ataxin-3, causes the symptoms of SCA3, there is still no treatment to stop the disease progression. As a monogenic disorder, SCA3, like other Spinocerebellar ataxias (SCA), is a promising candidate for gene therapy. While there are no approved gene therapies for SCA yet, there any several research labs and companies working towards achieving this goal.

The researchers in this study have been working on gene therapy for SCA3 since 2008. They have researched how gene therapy could offer protection against further decline, in several cell and mouse models of SCA3. They used an approach where they decreased the levels of the mutant Ataxin-3 gene while leaving the normal Ataxin-3 gene intact. This is known as allele-specific targeting. They demonstrated that using this technique, they could significantly reduce the behavioral and neuropathological changes that occur in SCA3 mice. Mice treated with the gene therapy showed improvements in their balance and motor coordination. 

Gene therapy in its most basic form involves two components, the gene that will replace or remove the diseased gene and a vector that will transport this new gene to its site of action. The most commonly used vectors today are adeno-associated virus (AAVs) followed by retrovirus. These viruses have been specifically engineered to deliver their passenger to the specified location. While both vectors have been through several years of preclinical and clinical testing for numerous gene therapy candidates, there are questions that remain regarding their safety. (1) Does the gene therapy product continue to be expressed in the targeted area long-term; (2) If there is long-term expression does it cause any adverse measurable effects to the targeted area; (3) Does the long-term expression affect the normal functioning of the targeted cells/organ.

In this current study, the researchers systematically tested the safety of lowering the levels of mutant Ataxin-3 specifically in the striatum of adult wild-type mice. The gene, a short hairpin RNA (shRNA) that lowers the level of the mutant Ataxin-3 protein, was packaged into a vector. The vector used in this study is a lentivirus. This is a type of retrovirus that infects non-dividing cells, like the neurons in the brain. Therefore, when the lentivirus vector is injected into the brain, it transports its shRNA cargo to the neurons.

As an experimental control, this study used both mice that were not injected and mice injected with an inert substance at the same two locations as the shRNA. Since wild-type mice do not express mutated ataxin-3, this study only looks at the long-term effects of expressing the lentivirus with the shRNA.

At three different time-points (2, 8, and 20 weeks after injection or delivery of the vector and its shRNA cargo) the mice were sacrificed. Then their brains were collected and analyzed. An important feature of any gene therapy product is its expression profile. This includes information like its tissue distribution to the duration of its expression. The shRNA against mutant Ataxin-3 contained a reporter. This reported would allow any cells with the shRNA to be identified at the end of the in-life study. The brains were sectioned in very thin slices so specific proteins in the cells could be labelled with the use of antibodies. At 2 weeks some cells expressed the reporter protein, with expression progressively increasing by 8 weeks and even more so at 20 weeks after gene delivery. This data showed long term and stable expression of the shRNA.

One concern in gene therapy is whether the long-term expression of the gene can induce unfavorable consequences to the cells in the brain. Using antibodies to label neuronal proteins, the authors found that while at 2 weeks after injection there was clearly loss of neurons at the injection site, by the later time points, this loss was no longer apparent. The study authors proposed that this recovery could be due to the birth of new neurons and/or the process of neuronal sprouting where neurons generate additional branches that make contact with neighboring neurons.

A major limiting factor for gene therapy is the host immune response, which is activated when the body sees the new vector as a foreign invader. In this study, the researchers looked at inflammatory signals in the brain. Microglia and astrocytes are two cell types in the brain that are activated upon injury and inflammation. Astrocytic and microglial activity increased soon after injection only in animals where the shRNA against mutant Ataxin-3 was injected.  By 8 and 20 weeks their levels returned to the levels seen in the non-injected mice. A special type of inflammatory proteins, known as cytokines, were elevated after injection but also returned to control levels by 20 weeks. Together, the results showed that even when inflammation was triggered early in the course of the therapy, it dissipated, eventually returning to control levels in the later time-points.

This was a carefully conducted study to evaluate the safety profile of a gene therapy candidate for SCA3. In their earlier proof of concept study, the authors showed that lowering the levels of mutant Ataxin-3 improved several abnormal features in SCA3 mouse models. This current study shows that the use of this therapeutic agent in wild type mice is safe up to 20 weeks after gene therapy delivery. While this study used a localized injection of the gene therapy agent only to the striatum, several brain regions are affected in SCA3. An additional study using a delivery route that targets multiple brain regions is necessary to evaluate the safety profile.

Future studies are necessary to characterize the expression profile and safety in non-human primates. The route of administration would be similar to that of human patients, which would allow the results to be more translatable for clinical trials. This is truly an exciting time for gene therapy, but it is also important to keep the safety of patients a top priority.  

Key Terms

Gene: A unit of heredity that contains our DNA, the code that controls the development and function of our body

Monogenic: A disorder or disease involved or controlled by a single gene

Vector: A mode of transport to carry foreign genetic material into another cell

RNA: A nucleic acid that carries instructions from DNA to make proteins. You can learn more about RNA here.

Short Hairpin RNA: A type of RNA folded into a hairpin structure that can target genes and silence them.

Conflict of Interest Statement

The author and editor declare no conflict of interest.

Citation of Article Reviewed

Nóbrega, C, et al. RNA interference therapy for Machado-Joseph Disease: Long-term safety profile of lentiviral vectors encoding short hairpin RNAs targeting mutant Ataxin-3. Human Gene Therapy, 2019. 30:7 https://doi.org/10.1089/hum.2018.157