Peptide conjugated oligonucleotides for splice switching therapy of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, arising from loss-of-function of the SMN1 gene. Mutations in SMN1 result in motor neuron degeneration, accompanied by peripheral manifestations including skeletal muscle atrophy. SMA is rare with an incidence of ~1:10,000 live births; however 1 in every 35 unaffected individuals is likely to be a carrier for the disease. Most affected SMA infants typically have a severe form of the disease with a mean life expectancy of ~2 years in age. However children with less severe disease can survive beyond 2 years but with poor quality of life. SMA severity relates directly to the level of functional SMN protein that a patient produces. A closely related gene to SMN1 is SMN2, although this gene typically only produces ~10% of fully functional SMN protein. However some SMA patients have additional copies of the SMN2 gene, and hence can produce more functional SMN protein. This mitigates disease severity and such patients typically have a milder disease course.

Most SMN2 gene product is not functional because the gene generates two distinct mRNAs via alternative splicing i.e. most of the mRNA lacks exon 7 and generates only partially functional protein. A HIGHLY NOVEL TREATMENT has been developed which acts on SMN2 mRNA to influence inclusion of exon 7 to generate functional SMN protein. This treatment utilizes splice switching oligonucleotides (SSOs), which are chemically modified DNA fragments that bind specifically to the SMN2 mRNA. SSO therapy has shown great potential in recent years for the disorder Duchenne muscular dystrophy (DMD) where they are now in advanced clinical trials. An SSO therapy has potential for even greater impact in SMA because it has potential to treat nearly 100% of affected SMA patients.

The major challenge to successful development of an SSO therapy for SMA is delivery of the SSO drug to critically affected tissues including motor neurons and skeletal muscle. Delivery to the spinal cord typically requires invasive methods, while targeting important peripheral tissues, such as skeletal muscle, is inefficient and often requires high SSO concentrations. The current state-of-the-art, led by Isis Pharmaceuticals, involves a clinical trial in which SSO is administered invasively to SMA patients via injection into the fluid surrounding the spinal cord and which therefore does not treat peripheral tissues.

Our aim is to develop an advanced SSO that can be administered non-invasively through a simple intravenous injection to treat peripheral tissues and also penetrates sufficiently well into the brain and spinal cord to treat the neurological component of the disease. We have developed advanced SSOs to which are coupled short protein fragments (peptides) to facilitate improved SSO delivery to peripheral tissues and provide penetration into the brain and spinal cord. A key initial step is to select the optimal peptide. Building on our existing peptide platform, we will also study a number of additional peptides with high brain delivery potential. Once a candidate peptide and SSO has been selected we will conduct extensive studies into the tissue distribution and tissue concentration of the SSO drug and also into the optimal dosing requirements. This information will allow us to design a multi-dose, long-term SSO therapy regimen to systemically treat all tissues associated with SMA, and we will evaluate this rigorously in an animal model of SMA. Our previous success with treating DMD and experience with peptide chemistry in relation to SSO drugs makes us uniquely well-placed for success in this project. Success will be followed by steps towards clinical development of the lead SSO drug.

SPINAL MUSCULAR ATROPHY (SMA) is a leading genetic cause of infant mortality due to progressive lower motor neuron death leading to muscle weakness. SMA is caused by loss of the SMN1 gene. Patients carry the very closely related gene SMN2 which is only partially functional due to its alternative splice exclusion of exon 7. There is a clear correlation between the variable copy number of SMN2 and the disease course, with milder patients having 3 or more SMN2 gene copies.

SPLICE SWITCHING OLIGONUCLEOTIDES (SSOs) are single-stranded, steric block oligonucleotides which, when directed toward splice sites of pre-mRNA, block the binding of the splice enhancing or silencing proteins. SSOs which modify SMN2 splicing to facilitate exon 7 inclusion and generate functional SMN protein are being tested in early Phase I/II clinical trials. Current SSOs do not penetrate the blood brain barrier (BBB) and are therefore administered through invasive intrathecal injection. This is necessary for adequate spinal cord drug delivery but is not practical as a long term therapy and also fails to treat systemic features of the disease, especially important in the most severe cases. We have developed advanced SSOs comprising a peptide-conjugated SSO platform permitting effective systemic SSO delivery to peripheral tissues (e.g. skeletal muscle) and brain penetration which we will now take through rigorous preclinical development. Utilizing the PMO SSO chemistry, we will optimise PMO length and sequence for modifying SMN2 splicing and test three lead peptides for highest level of delivery to peripheral tissues and spinal cord. The most effective peptide-PMO compounds will be extensively studied to determine pharmacokinetics and an optimal treatment dose in order to generate a multi-dose, long-term treatment regimen that will be studied for efficacy and safety in SMA model mice. This lead compound will subsequently be taken forward for clinical development to be evaluated in SMA patients.

The present project aims to develop novel, advanced generation oligonucleotides for therapeutic application in Spinal Muscular Atrophy (SMA). There thus is likely to be high impact with numerous individuals, groups and organisations likely to gain significant benefit:

ACADEMIC BENEFICIARIES will include the PI, the Project Team, the PI's and Co-I's UK collaborators (e.g. the MDEX Consortium) and non-UK international collaborators. Other academic beneficiaries will include groups working in the SMA and related neuromuscular, motor neuron and neurodegenerative disease fields, the oligonucleotide therapeutics field, and in relation to macromolecular drug delivery to the brain.

NON-ACADEMIC BENEFICIARIES will include SMA patients, their families and charitable organisations that support SMA patients and research, and those in the biotechnology, pharmaceutical and investment sectors that have interests in relation to SMA, oligonucleotide therapeutics and to the therapy of rare diseases more generally.

ACADEMIC BENEFICIARIES will benefit in the short term from direct knowledge outputs including publications and other forms of research communication. Knowledge gained will inform future research directions, future funding applications, future research ideas and projects, and future collaborative opportunities. For example, knowledge gained in relation to specific peptides with utility for targeted delivery of oligonucleotides will provide insight into understanding of mechanisms (leading to future improvements), utility for application to other diseases (e.g. other neurological diseases) and to delivery of other drug cargoes (e.g. therapeutic siRNAs, miRNAs). Such specific knowledge and similar gains in project knowledge will not be restricted to the PI and Co-Is although these groups are likely to benefit soonest. Longer-term, success in numerous aspects of this project will provide fundamental knowledge in relation to the treatment of neurological disorders and will likely lead directly and indirectly to numerous long term academic beneficiaries building on and exploiting this knowledge in relation to other common and rare neurodegenerative diseases in particular and neurological diseases more generally.

NON-ACADEMIC BENEFICIARIES especially SMA patients will benefit directly from an advanced oligonucleotide therapy, which could be available for clinical testing in approximately 5 years and offer disease modification and improved quality of life. SMA families will benefit immediately because such research is crucial to maintaining the morale of the SMA community. In the medium term, families of SMA patients will benefit directly from a therapeutic agent (i.e. a product) that can be tested on their children offering the prospect of disease modification. SMA charities and foundations play a crucial role in supporting families and funding early stage research. Such organisations will benefit from encouraging results as they play a key role in supporting, informing and motivating SMA patients and their families. In the medium term such organisations will benefit as they play a critical stakeholder role during clinical testing and in guiding the development and application of a new therapy, including in relation to research, ethical and commercial issues. Finally, non-academic beneficiaries in the commercial sector are likely to benefit directly in the medium term through opportunities to acquire licences to intellectual property in relation to aspects of the technology - either specifically in relation to clinical development of the lead compound for SMA or more generally in relation to platform peptide and/or oligonucleotide technology that could have broad application. This is likely to lead to further development, a range of possible products and a range of possible commercial benefits for companies in the biotechnology or pharmaceutical sectors and/or investors in relation to such companies.