As a team based at a university with one of the largest faculties of medical and life sciences in the world, we have a strong dedication to transforming lives through medical innovation. Additionally, all of our team members come from a life sciences background and felt motivated to hone in on the applications of synthetic biology to therapeutics. Thus, we sought out gaps in the medical field, looking for areas that require novelty. After a little research, we found that rare genetic diseases and their current treatments required innovation and consideration. The motto of Guy’s Hospital, which is where we are based, is “dare quam accipere”, or “to give rather than receive”. These words resonated with us as we decided to focus in on rare genetic diseases, which are a considerable cause of suffering and premature mortality.

Why Rare Genetic Diseases?

Rare genetic diseases are those which have an incidence of less than 1 in 2,000 [5]. There are approximately 6,000 diseases that fit into this category. Consequently, although rare genetic disorders have low individual incidence, they are as a whole common worldwide. The statistics are extremely grim. In the UK alone, 1 in 17 people will be affected by a rare genetic disorder, which amounts to about 3.5 million individuals. This is comparable to the number of UK citizens affected by diabetes, which is approximately 4 million individuals [5]. However, there are a wide array of treatments available for diabetes patients. This starkly contrasts the fact that only 6% of rare genetic disorders have available treatments [3]. Moreover, two-thirds of these congenital diseases are serious and disabling, three-fourths affect children, and nearly half are life limiting [3]. Considering these statistics one would assume that more funding, research, and specialisation has gone into the treatment of these disorders. Unfortunately, the system is geared towards the development of therapies for more common genetic disorders, due to the fact that there are very small population sizes to carry out clinical trials with. Furthermore, few clinicians specialise in such disorders due to the small quantity of patients. This, combined with the hesitancy to invest in orphan drugs by stakeholders and research facilities, has made developing therapeutics for rare genetic diseases difficult, as shown in the fact that only 1% of the treatments available are curative [3]. The general lack of treatment for these diseases motivated us to fill this gap, and to aim to provide a universal solution for the millions affected by such congenital disorders.

We also found that there is limited access to genetic services with regard to rare genetic diseases. These include diagnostic, therapeutics, and counselling services for individuals suffering from these disorders or their families. National policy, legal status, and the local clinicians all play a crucial role in determining the information given to parents regarding their options, whether it be terminating the pregnancy, potential to take part in clinical trials, or which treatment to be carried out. Many countries genetic services integrated into their specialist services, meaning only a select portion of the population has access to the fundamental information and support needed when faced with a potentially fatal disease [2]. Consequently, we wanted to ensure to create materials that would help families with rare genetic diseases and provide them with the information they rightly deserve. We included this in the human practices and public engagement aspects of our project.

The final characteristic of rare genetic diseases that intrigued us is their potential to be cured with gene therapy. 80% of rare genetic diseases are due to single gene mutations, thus making them prime candidates for gene therapy [2]. Despite the great diversity amongst these disorders in the clinical setting, single gene disorders all have a common biological origin and and need the same genetic management. Thus, their uniform need for gene replacement, which can be carried out by gene therapy, makes them excellent candidates for this technology [2].

Gene Therapy: A Universal Solution?

Gene therapy presents a plausible curative solution to a wide array of genetic diseases First conceived in the 1970’s, this approach was offered as a revolutionary solution to monogenic and hereditary disorders. Today, however, this technology is used for a wide array of diseases, from cancer to Alzheimer’s. This is because the gene therapy is based on the administration of genetic material into cells to neutralise the effect of unhealthy genes via the production of the healthy protein product. There are two main routes by which this is done; ex vivo and in vivo administration routes. The former relies on the extraction of cells from the individual, which are then modified and transplanted back into the individual. While this procedure protects against an adverse immune effects, it is too personalised and limits the number of patients that can be effectively treated. As time equals lives, we decided to focus on the latter method of gene therapy.

Figure 2: Flow chart demonstrating the process of gene therapy; adapted from Ramamoorth and Narvekar.

In vivo gene therapy involves the delivery of the therapeutic gene material and its regulatory elements into the cells of interest. Vectors are carriers that are used to bring the therapeutic gene to the target cells. Roughly, vectors can be categorised as viral and non-viral vectors. Nonetheless, once the vectors have reached the target cells, they genetic material must become integrated with the nucleus and replaces the mutated gene. Consequently, the therapeutic gene corrects the loss of function mutation or compensates for the hypomorphic mutations. The most crucial step in this form of gene therapy is the selection of the appropriate vector. However, once optimised, this method of delivery can be applied to the entire population of individuals with the disease. We found this particularly appealing in our focus on rare genetic diseases, as the optimization of a single vector could be applied to the vast majority of these diseases. As it is impractical to create personalised solutions for each disease, we decided to focus on improving the vectors available for gene therapy for rare genetic diseases. This requires a look into the two kinds of vectors:

Viral Gene Therapy:

Viral vectors are currently the most researched form of vector for gene therapy. Viruses function via the infection of cells with their genome, which is followed by the replication of the viral genome and the construction of new viral particles. In viral gene therapy, the ability of viruses to infect cells is exploited. Furthermore, viral gene therapy takes advantage of the spatial seperation of the cis-acting regulatory sequences and the trans-acting viral coding genes. In designing a viral vector, the viral genome is removed and replaced with the therapeutic gene. The regulatory sequences for this gene also replace the regulatory sequences of the virus. This allows for transduction in the place of infection. Transduction is the process by which the virus “infects” the cell with the therapeutic gene and is unable to carry out viral assembly. In addition to the removal of the viral genome, a great deal of the pathogenic properties of the virus are removed. This allows for an effective and potent method of gene transfer, and there are several approved viral gene therapies on the market, especially for monogenic disorders. However, that does not mean that viral gene therapy is not without faults. Immunotoxicity is a significant problem, as the immune system does recognise the antigens on some vectors; this lead to the first fatality in response to viral gene therapy. Furthermore, there is the issue of specificity and dosage; the vector must be able to bring the therapeutic gene to the correct target cell in the proper dosage to prevent acute toxicity and to ensure effectiveness. Additionally, viral vectors are limited by size; they have a limited packaging capacity; most viral vectors can only carry 6-8kb. These limitations pose great challenges for those developing viral gene therapies.

Non-Viral Gene Therapy:

Non viral gene therapy involves the delivery of therapeutic gene in three main methods; Naked DNA, particle based, and chemical based. This form of delivery is considered to have greater bio safety in comparison to viral vectors, as the inflammatory responses and insertional mutagenesis that can arise are avoided. Low pathogenicity and low cost of production has made non-viral vectors a prime subject for researchers across the world. Generally, non viral vectors are used in the transport of nucleic acids. These include small DNAs, large DNAs, or RNA. These nucleic acids then act to modify gene expression in a beneficial way either by transcription or interference. We are particularly interested in RNA interference (RNAi) or RNA silencing is an umbrella term that describes the post-transcriptional gene silencing carried out by small RNA molecules, especially siRNAs. Since their discovery, siRNAs have been seen as an excellent candidate for therapeutics in which aberrant genes need to be silenced to achieve a beneficial result. They work by inhibiting the translation of the mRNA transcript with the complementary sequence. siRNAs bind to the mRNA transcript and induces its cleavage, thus categorising the mRNA as abnormal leading to its degradation. Consequently, the protein product is downregulated. Even so, the primary issue with using RNAi as a therapy is the efficiency of the non-viral delivery mechanisms.

During our interview with Dr Williams, who specialises in neuronal ceroid lipofuscinosis or Batten’s disease, it was made clear to us that gene therapy is a current solution to a number of rare genetic disorders. This was extended by our interview with Dr Jungbluth, who runs clinics for children with neuromuscular and neurodegenerative disorders. Dr Jungbluth informed us of the limitations of current gene therapy regarding neuromuscular disorders. Gene therapy is not readily available for a number of neuromuscular disorders as the therapeutic gene is too large for the vector. This is primarily because the packaging capacity of viral vectors is insufficient to host the therapeutic gene and thus carry out transduction. This can be applied to a wide variety of not only rare genetic disorders but many of the common single-gene disorders. Consequently, it became our aim to investigate the current limitations in viral vector vector capacity and to increase their potential to accommodate larger genes [7].

Defining the Problem:

Rare genetic diseases are difficult to work with and thus historically did not have as much attention as they should, resulting in only 6% of treatments for approximately 6,000 diseases that affect 3.5 million in the UK and approximately 400 million worldwide.
There is a lack of available information and guidance for those suffering from rare genetic diseases.
A plausible and curative solution to a wide variety of rare, single-gene disorders.
The most common and effective form of gene therapy for rare, single-gene disorders is the in vivo administration of viral vectors with the therapeutic gene, which will compensate for the unhealthy gene by producing the healthy protein product.
However, many viral vectors are limited by their packaging capacity, thus vectors must be able to carry therapeutic genes of larger sizes to increase the number of rare genetic diseases that can be treated using gene therapy.
RNA interference is a mechanism by which gene expression can be suppressed, which is ideal for dominant disorders. Manipulation of small interfering RNAs, which carry out RNA interference could be used to control gene expression.
Non-viral vectors serve as an alternative to viral vectors, yet they are limited by their delivery efficiency.

Our Solution:

RNA-mediated regulation of gene expression.

Following our discussion with Professor Heinz Jungbluth, we decided to focus on the problem of packaging capacity and gene therapy. To provide a solution to this problem, we looked to RNA interference. As described prior, RNA interference is a mechanism by which gene expression is inhibited using small non-coding RNAs. Synthetic small RNAs can be used to control and manipulate gene expression. We have decided to do this in bacteria, specifically E.coli to show that we can regulate the expression of protein capsid proteins to construct viruses with larger packaging capacities. The equivalent of siRNAs in bacteria are known as small RNAs. These inhibit gene expression in a way that is similar to that of siRNAs in mammalian cells. However, they may or may not show absolute complementarity to the mRNA transcript, unlike siRNAs. sRNAs are typically trans-encoded, thus show a degree of complementarity to the mRNA transcript, leading to the formation of incomplete Watson-Crick interactions between the two. The majority of trans-encoded sRNAs bind to the ribosomal binding site (RBS), thus interfering with the interaction between the mRNA and the 30S ribosomal unit. They may also bind upstream of the start codon. Regardless, the sRNA and mRNA duplex are degraded via the enzyme RNase E. As no protein product is produced, the gene is down-regulated. We have decided to use three different sRNAs to demonstrate their ability to down-regulate gene expression to ultimately be used to manipulate the ratios of viral capsid proteins. This will additionally address the use of small RNAs as a form of gene therapy themselves.

Application to Synthetic Biology:

Synthetic biology application involves the design and creation of biological components that do not already exist in the natural world. Our project aims at the creation of novel sRNA BioBricks to artificially regulate gene expression. The applications of our BioBricks are two-fold; they have the potential to serve as therapy for rare genetic disorders via RNA interference as well as in the expression of viral capsid proteins for the construction of novel viral geometries. Our project thus perfectly coincides with the ethos of synthetic biology, as it has the capacity to develop non-existing viral capsids with novel features.

References:

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Boycott, Kym M., and Diego Ardigó. “Addressing Challenges in the Diagnosis and Treatment of Rare Genetic Diseases.” Nature Reviews Drug Discovery, vol. 17, no. 3, 2017, pp. 151–152., doi:10.1038/nrd.2017.246.
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Mali, Shrikant. “Delivery Systems for Gene Therapy.” Indian Journal of Human Genetics, vol. 19, no. 1, 19 Jan. 2013, p. 3., doi:10.4103/0971-6866.112870.

Ramamoorth, Murali. “Non Viral Vectors in Gene Therapy- An Overview.” Journal Of Clinical And Diagnostic Research, Jan. 2015, doi:10.7860/jcdr/2015/10443.5394.

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Shahryari, Alireza, et al. “Development and Clinical Translation of Approved Gene Therapy Products for Genetic Disorders.” Frontiers in Genetics, vol. 10, 2019, doi:10.3389/fgene.2019.00868.

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Team:KCL UK/Description

Project Description

Abstract:

Inspiration:

Dare Quam Accipere: Our focus on medical innovation