The Ins and Outs of Antisense Drugs

D RUG D ISCOVERY FALL 2013

CA PublicOation DedicaMted To ResearchP UpdateAs SS Antisense Therapeutic Approach for SMA The Ins and Outs of Antisense Drugs

ABC’s of antisense drugs Antisense drugs are small snippets of synthetic genetic material that bind to RNA (short for ribonucleic acid ). The main factor driving where the antisense drug binds How does the antisense to the RNA is the nucleic acid sequence used. Nucleic acids consist of a chain of therapeutic approach work linked units called nucleotides. Antisense drugs are typically 12-21 nucleotides that for SMA? are chemically modified to bring about the appropriate drug response. (Figure 1) - In SMA, the survival motor neuron (SMN) protein level is reduced due to How does the antisense approach work for SMA? the loss of the SMN1 gene. The low SMA is caused by a loss of, or defect in, the survival motor neuron 1 (SMN1) gene. level of this protein causes the motor The SMN1 gene produces most of the SMN protein, which is critical to the health neuron nerves to not function correctly. and survival of the nerve cells in the spinal cord responsible for muscle function. - A second closely related gene called The severity of SMA is correlated with the amount of SMN protein in the cell. A SMN2 – the back-up gene - exists that second closely related back-up gene called SMN2 exists that normally produces a normally produces a shorter, low- truncated, low-functioning form of SMN protein. functioning form of the SMN protein. Antisense therapy could be used to change the functioning of the SMN2 gene by utilizing alternative RNA splicing. Alternative splicing is a normal mechanism that - Antisense drugs are small snippets of the cell uses to produce different, but closely related proteins from a single gene. synthetic genetic material or RNA that utilize a natural RNA processing How do antisense drugs cause the SMN2 gene to produce mechanism to increase the production of the SMN protein. more functional SMN protein? To understand alternative splicing, it's important to understand how DNA creates - It is called “anti-sense” technology, messenger RNA (mRNA). because the pieces of synthetic DNA carries the genetic information of a cell and consists of thousands of genes. material run in a “mirror-image” or Each gene serves as a recipe on how to build a protein molecule. mRNA is created “reverse” or “anti” order from the when the entire DNA strand is copied. This includes the sequences that encode for natural RNA that it binds to. proteins. and regions that are unnecessary for making proteins. Before mRNA can - Antisense drugs can be designed to function, the regions that are unnecessary for making proteins are deleted from the bind to very specific regions of the RNA strand. SMN2 back-up gene to enhance the production of the full length, fully- functioning SMN protein. Figure 1. This figure shows the structure of the DNA double helix through Watson-Crick - This increase in the amount of nucleotide base pairing. It is comprised of four different types of building blocks normal SMN protein produced by called nucleotides. These are designated A, T, C, and G. the SMN2 gene should provide the Note that in DNA, nucleotide adenine (A) in a DNA necessary amounts of SMN protein molecule always pairs with the nucleotide thymine (T), needed for proper muscle function while cytosine (C) always pairs with guanine (G). and development.

continued on next page The Ins and Outs of Antisense Drugs.

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The natural process that removes these regions and re-forms the finished mRNA is called “splicing.” Through the splicing process, the cell can create many diverse proteins from a single gene and splicing accounts for most of the diversity in proteins in the cell. In fact, of the approximately 25,000 genes in the human genome, approximately 90% have alternative splice forms. Antisense drugs can be designed to bind specific regions of the RNA and change the splicing of the SMN2 gene to increase the production of functional SMN protein. The antisense drug binds and promotes the inclusion of a specific nucleotide sequence (exon 7), correct - ing the known pre-mRNA processing defect in splicing in the SMN2 gene. This allows the SMN2 back-up gene to express more full-length SMN protein, rather than a truncated, low-function - Figure 2. This figure shows splicing of RNA and where the antisense drug ing protein. changes the splicing to produce full-length SMN protein.

What exactly does the antisense drug do at the molecular level to the SMN2 gene? Normally SMN2’s mRNA undergoes incorrect splicing and encodes for an unstable SMN protein, known as 7SMN. The production unstable SMN pΔrotein is the result of exon 7 being skipped during the splicing process of the SMN2 pre-mRNA (Figure 2). The clinical result is low amounts of full length SMN protein and ultimately decreased motor function in SMA patients. The use of anti-sense oligonu - cleotides, targeting sequences in the SMN2 pre-mRNA, can correct the spicing defect and promote exon 7 inclusion, leading to higher SMN protein levels.

Influence of backbone chemistry Figure 3. Nucleotide analogues used in antisense oligonucleotide drugs. A secondary factor, in addition to the Structures of various nucleotides or nucleotide analogues commonly used in nucleic acid sequence, that influences antisense drugs are shown. The antisense oligonucleotide developed by the ISIS the effectiveness of an antisense drug is group to improve SMN2 splicing has the 2 -MOE modification (red). The one being the backbone chemistry used to hold used by the Burghes group is called a Morp′ holino and is seen in the bottom left. From Rigo et al., J Cell Biol. (2012) 199(1):21-5. the nucleotides together. See Figure 3. The different chemistry can influence Compass 20 13 www.curesma.org many factors, including: 1) how the Isis-SMN Rx Program Update on drug distributes throughout the body; 2) the length of time the drug persists in Ongoing and Future Clinical Trials a given tissue; 3) the efficiency of drug uptake into cells; 4) how strongly the drug binds to the target RNA; and 5) Infant Program Update: Child (Type 2 and Type 3) the likelihood of non-specific interac - Isis Opens Cohort 2 of the Program Update: tions with other RNAs. Clinical Study in Infants Isis Completed Dosing and with SMA Enrollment in the Multi- Drugs for SMA need to cross This study is evaluating the investiga - Dose Phase 2 Dose the blood brain barrier tional compound, ISIS-SMN Rx , in Escalation Study in Children For a treatment for SMA to be effective infants with Type 1 Spinal Muscular with SMA a drug has to be active in motor Atrophy (SMA). This Phase 2 study is This Phase 2 study is a multiple-dose, neurons and other cell types in the an open-label, multiple-dose, dose- dose-escalation study designed to central nervous system (CNS). This will escalation pilot study, designed to examine the safety and tolerability of require the drug to be present in examine the safety and tolerability of multiple doses of the drug given over a cerebrospinal tissues. ISIS-SMN Rx in infants with SMA and longer period of time. This study is to provide data to define the optimal Antisense drugs do not cross an intact being conducted in children age 2–15 dose for future studies in infants. To blood–brain barrier, but there are with Type II and Type III SMA. The meet enrollment criteria, infants must several approved methods and devices study is fully enrolled, has completed be between the ages of three weeks and available for delivery of drugs into the dosing, with data expected once the seven months, live in close proximity cerebrospinal fluid including direct follow up is completed early next year. to a study site and pass additional injection into the intrathecal space. screening evaluations conducted at the Antisense drug injected in this manner study site. The study is being con - Next Steps: distributes broadly into CNS tissues. ducted at 4 centers in the United States A larger controlled Phase 3 study in Intrathecal delivery refers to the and Canada who are looking for children is planned to begin in 2014. administration of a drug directly into eligible subjects - This study will be larger than our the cerebral spinal fluid (CSF), a fluid • Stanford University Medical Center current ongoing study and will provide that surrounds the brain and spinal in Stanford, California more opportunity for enrollment at an cord tissue. The drug is administered increased number of participating • Nemours Children's Hospital in through an injection in the lower back sites, including Canada and other Orlando, Florida into a fluid-filled space below the end of international sites. the spinal cord, resulting in broad • Columbia University Medical If you would like further informa - distribution of the drug in various brain Center in New York, New York tion, please contact your study site or regions and the spinal cord. This is a • The Hospital for Sick Children you can contact Isis directly at: common procedure in hospitals (SickKids) in Toronto, Ontario, Kristina Lemonidis frequently used for pain relief. Canada ([email protected]). For further study information and contact information for the study Please go to sites, please visit .gov www.clinicaltrials www.clinicaltrials.gov and search for ating in for info on particip ISIS-SMN Rx . cal trials. ongoing SMA clini Next Steps: A controlled Phase 3 study in infants is planned to begin early in 2014. This study will be larger than the current ongoing study and will be conducted in the US and internationally.

Figure 4. Illustration of intrathecal delivery.

Compass 20 13 www.curesma.org New FSMA Research Funding

Families of SMA Awards $150,000 to Dr. Hua in the Laboratory of Dr. Adrian Krainer at CSHL to Assess the Influence of Backbone Chemistry on Antisense Drugs for SMA.

This is the fourth drug discovery project funded by Families of “As a Research Investigator in my lab at SMA in 2013 with a total investment of $550,000. This pro - Cold Spring Harbor Laboratory, and as gram will systematically assess the effect of backbone chemistry part of our long-standing collaboration on the therapeutic efficacy of Antisense Oligonucleotides with Isis, Dr. Hua has been doing pioneer - (ASO) that target the ISS-N1 region of the SMN2 RNA. ASOs ing work towards the development and that bind to the ISS-N1 region will be compared. This is the characterization of the mode of action of binding region of the drug ISIS-SMN Rx that is currently being ISIS-SMN Rx . I am grateful for the support tested by Isis Pharmaceuticals in SMA clinical trials. This from Families of SMA, which will allow funding is being awarded to Dr. Yimin Hua in the laboratory of Dr. Hua to study key aspects of ASO Dr. Adrian Krainer at Cold Spring Harbor Laboratory. Both efficacy in SMA mouse models, with scientists are long-term collaborators with Isis Pharmaceuticals relevance to the clinic,” said Dr. Adrian Krainer, Ph.D., and actively participated in the pre-clinical development and Professor at Cold Spring Harbor Laboratory. characterization of the mechanism of action of ISIS-SMN Rx . “ASO10-27 ( ISIS-SMN Rx ) and its longer derivatives, which restore SMN expression Program Description: through correction of SMN2 splicing, hold Comparison of efficacy mechanisms of MOE and PMO great promise to treat spinal muscular antisense splicing therapy in type I SMA mice. atrophy. ASOs must be chemically modi - fied to enhance their stability and facilitate Objective: The project goal is to compare the ISS-N1 antisense their binding to the target RNA. ASOs oligonucleotides using two alternate backbone chemistries with different chemical modifications called MOE and Morpholino (PMO). Please see the first article have been tested in various SMA mouse in this edition of Compass for more information on backbone models; two types of modifications, MOE chemistry of ASOs. Both chemistries have shown great promise and morpholino, showed striking improvement including in treating SMA by correcting SMN2 splicing. In this project, long-term survival. However, there were apparent differences the team will systematically explore parameters that influence among various studies, in terms of optimal ASO dose and the efficacy of each type of ASO. sites of delivery. We will conduct a comprehensive side-by- Research Strategy: We will use a severe SMA mouse model to side comparison of MOE and morpholino ASOs in a severe compare the therapeutic effects of antisense oligonucleotides mouse model, and study the mechanisms underlying any with two types of chemical backbones, delivered either to the observed differences in ASO potency and efficacy,” said Yimin central nervous system or under the skin. We will then perform Hua, Ph.D., Research Investigator at Cold Spring Harbor detailed pharmacological studies to compare their distribution Laboratory. and efficacy in different tissues and cell types. This project will provide important information for further developing the “We have a long term collaboration with Drs. Adrian Krainer and Yimin Hua to identify an antisense drug to treat spinal antisense oligonucleotide drug, ISIS-SMN Rx , an antisense oligonucleotide drug candidate that is currently in clinical trials muscular atrophy. This has been a very productive collabora - by Isis Pharmaceuticals. This project may also help understand tion resulting in the identification of ISIS-SMN Rx , which is which tissues or cells are most important in SMA pathogenesis currently in clinical trials. We would be delighted to continue in this mouse model. to work with Dr. Hua to investigate the mechanistic differ - ences between the different olignucleotide chemistries. Dr. Hua has a strong track record of successfully completing projects and making important contributions to our under - standing of SMA and antisense drugs,” said Dr. C. Frank Bennett, Ph.D., Senior Vice President of Research, Isis Pharma - ceuticals.

Compass 2013 www.curesma.org Families of SMA Awards $150,000 to Dr. Burghes and Dr. Lorson to Investigate New Antisense Therapies for Spinal Muscular Atrophy.

Families of SMA is dedicated to creating a treatment and cure “There are now a series of exciting for Spinal Muscular Atrophy by funding and advancing a therapies for induction of SMN from the comprehensive research program, including drug discovery back-up gene SMN2, with the antisense programs to make practical new therapies. This new program oligonucleotides being one. In essence, will assess the therapeutic potential of second-generation the best way to increase SMN protein Antisense Oligonucleotides (ASO) sequences for SMA. This from SMN2 remains a critical issue, and funding is being awarded to Co-Principal Investigators Dr. this will be investigated in this new Arthur Burghes at Ohio State University and Dr. Christian project,” stated Arthur Burghes Ph.D., Lorson at University of Missouri. Professor at Ohio State University.

Program Description: “We are very grateful to Families of SMA New Morpholino Antisense Oligonucleotides for for funding our ASO project as we hope the Treatment of SMA. we can continue to develop therapeutics Objective: Antisense oligonucleotides (ASOs) targeted to the that target an additional genetic element ISS-N1 region have been shown to be effective in mice and are within SMN2. SMA is a very complex now being tested in the current clinical trials conducted by Isis disease and it is possible that more Pharmaceuticals. The major goal of this new program is to therapeutic options will be required to explore the therapeutic potential of two new sequences, which effectively combat this disease,” stated are located in different regions of the SMN2 gene. Importantly, Chris Lorson, Ph.D., Professor at Univer - the possibility exists to use these different ASOs in combina - sity of Missouri. tion. The ASOs in this project will be generated using a A portion of this new research program is being generously different morpholino backbone chemistry. funded by Stop SMA. Stop SMA was created to raise aware - Research Strategy: First, the Burghes and Lorson groups will ness and funding for research to treat and end SMA. determine the optimal nucleic acid sequence. They will then http://www.stopsma.org establish the best dose of the optimized ASOs to provide maximal rescue in the severe Delta7 mouse model of SMA, developed by Dr. Burghes. Knowing this information in mice will help design subsequent studies in primates to assess Families of SMA Funding Strategy whether these ASOs reach the correct tissues including motor and the SMA Drug Pipeline neurons, when delivered into the cerebral spinal fluid (CSF) in larger animals. Dr. Ravindra Singh at Iowa State University will There are now 15 new SMA drug programs in be collaborating on the project. development, including 3 in clinical trials. This pipeline has expanded from just 4 programs 5 years ago. Families of SMA has funded about SUPPORT OUR MS two thirds of all the ongoing drug programs for SEARCH PROGRA RE ma.org SMA. The FSMA research approach funds Please visit cures tion. programs at early stages, and then partners and make a dona with companies to take them through clinical trials. Supporting multiple programs gives different approaches for a SMA therapy, which increases the chances of success and acceler - ates the timeline to a treatment and cure. In 2013 alone, FSMA has funded four new drug This newsletter has been produced in a collaboration programs. See our website at www.curesma.org between Families of SMA, Biogen Idec, and Isis for the 2013 SMA Drug Pipeline. Pharmaceuticals.

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