Information for Patients Information for Physicians, Scientists and Investors Clinical Trials

Duchenne Muscular Dystrophy

AVI’s lead drug candidate, AVI-4658, is in clinical development for Duchenne Muscular Dystrophy.

Information for Patients

DMD is an incurable muscle–wasting disease associated with errors in the gene that makes dystrophin, a protein that plays a key role in the way muscles work. Our drug candidate AVI–4658 is in clinical testing and has been designed to skip exon 51 of the dystrophin gene, allowing for restoration of the information in the mutated gene needed to make dystrophin from mRNA. AVI believes that restoring dystrophin might improve, stabilize or significantly slow the disease process, and might prolong and improve the quality of life for specific patients with DMD.

AVI is also evaluating a drug candidate AVI-5038, designed to skip exon 50, which is in preclinical development.

Mutations that could be potentially corrected by skipping exons 51and 50 are as follows:

Exon to be skipped	Drug Candidate	Stage	Treatable Deletions
51	AVI-4658	Clinical Development Phase 1b/2 (UK) Preclinical Development stage (US)	45-50, 47-50, 48-50, 49-50, 50, 52
50	AVI-5038	Preclinical Development (US)	51, 51-53, 51-55

AVI BioPharma was granted orphan drug designation for AVI–4658 by the U.S. Food and Drug Administration (FDA) in November 2007 and by the European Medicines Agency (EMEA) in December 2008. AVI also received fast track status for AVI-4658 from the FDA in December 2007.

Because of the specificity of each drug, different errors in the dystrophin gene may require different oligonucleotide drugs to repair the error — the relationship between the type of error and type of repair is explained in the section entitled Information about Duchenne Muscular Dystrophy (below). Approximately 80% of all DMD patients could potentially be treated with exon–skipping drugs, but this would require more drugs than those in the current AVI pipeline.

Information about Duchenne Muscular Dystrophy

Dystrophin protein is essential for the function of muscles. Muscle fibers stretch and contract with great force when a muscle is used. Dystrophin acts as a spring and a shock absorber between the muscle fiber surface and its internal motor made up of protein called actin. In Duchenne muscular dystrophy (DMD), dystrophin is missing and as the actin motor causes the muscle to contract it damages the muscle fiber’s surface membrane because the force-absorbing protection of dystrophin is missing. Fortunately, muscle fibers can repair themselves. In small children with DMD, the force of muscle contraction is weak, there is not much damage and so repair can stay ahead of muscle damage. As children with DMD grow, their muscle strength increases and, eventually, muscle damage happens faster than it can be repaired. Permanent muscle damage becomes more and more widespread and eventually becomes life threatening as the vital muscles for breathing (diaphragm) and the blood circulation (heart) are affected.

The dystrophin protein is made from information in the dystrophin gene. It is the largest gene in the human body and contains 2.4 million pieces of genetic information (DNA). When cells divide, the genetic information stored in the gene must be copied perfectly and this almost always happens successfully. Even if an error is made, cells have machinery that can repair the mistake. Very rarely an error escapes detection and repair, the newly copied dystrophin gene is defective and that defect can be passed on from parent to child or can arise spontaneously in an affected child without any family history.

The most common defects in the dystrophin gene leading to DMD are so called deletions, meaning that a piece of DNA from the blueprint to make dystrophin protein is lost. The genetic change leading to Duchenne muscular dystrophy arises when a small fragment of DNA is lost in a particular part of the gene so that it ruins the remaining information in the gene and the cell cannot even use the remaining part of the dystrophin blueprint. At other times loss of the small fragment happens in places in the gene where the remaining information is not ruined and the cell has most of the blueprint to make dystrophin. In this case, although the dystrophin protein is missing a small piece it can still perform some of the shock-absorbing tasks of dystrophin and the patient has the milder, Becker form of muscular dystrophy (BMD).

Several years ago scientists found a way to restore the remaining part of the blueprint in the damaged dystrophin gene in DMD patient’s cells by a process called exon skipping and so make a shortened version of the protein which might still perform some of the shock-absorbing tasks of full length dystrophin. AVI BioPharma is now developing drugs that might ultimately restore the ability of the gene to make a shorter but working form of dystrophin in certain Duchenne muscular dystrophy patients. If these drugs are successful the course of the DMD might be slowed down and the severity of the muscle disease might be reduced.

The relationship between exon skipping and the DMD deletions is shown below for some of the more frequent deletions in DMD

Exon skipped	Repaired Deletions
51	45-50, 47-50, 48-50, 49-50, 50, 52
50	51, 51-53, 51-55
45	12-44, 18-44, 44, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55
53	10-52, 45-52, 46-52, 47-52, 48-52, 49-52, 50-52, 52
44	10-43, 19-43, 30-43, 35-43, 36-43, 40-43, 42-43, 45, 45-54
8	4-7, 5-7, 6-7, 3-7,
55	47-54, 48-54, 49-54, 50-54, 52-54, 54, 56, 56-62
7	2-6,8-11, 8-17, 8-43, 8-45
52	53, 53-55, 53-57, 53-59, 53-60
17	12-16, 18, 18-20, 18-22, 18-25, 18-27, 18-29, 18-33, 18-36, 18-38, 18-41, 18-44

AVI is currently testing only two candidate drugs, AVI-4658 and AVI-5038, which have been designed to skip exons 51 and 50 respectively.

Drug Candidates in clinical trials:

In January 2009, AVI announced results from a Phase 1 trial of AVI–4658. (Read press release) Biopsy data showed that injection of the drug into a foot muscle in a series of DMD patients significantly increased the amount of dystrophin in the muscle compared to the placebo treated muscle in the patient’s other foot. The amount of dystrophin expressed was related to the amount of drug injected, the drug was well tolerated and there were no significant drug–related serious adverse events.

A multi–dose, dose escalation trial to examine the efficacy of AVI–4658 following systemic administration (IV) was opened in December 2008 and the first patient was treated on February 18, 2009 (Read Press Release). This trial is being conducted in the United Kingdom. This study is ongoing with several boys now completing their 12 weekly injections. AVI is carefully monitoring how well the PMO is tolerated by the boys; and how effective each of the doses will be at producing some dystrophin in the biceps muscle of the arm. Full results cannot be released until the last boy has finished the complete study (including a lengthy follow up period) but initial data from the trial demonstrated corrected RNA and protein expression (Read Press Release). Plans are being drawn up for subsequent studies in Europe and an initial study in the US. These plans will need vetting and approving by the appropriate Regulatory Authorities.

In addition, AVI has now started the comprehensive series of preclinical tests necessary before a US study with AVI-4658 can be undertaken. The results from these studies will need to be reviewed by the FDA before a US study can start. It is hoped that the US study could start in early 2010.

AVI is advancing a second product, targeting exon 50, and has now started a similar series of preclinical studies with AVI-5038. While these studies are underway, AVI anticipates having discussions with the Regulatory Authorities about plans to start clinical work with AVI-5038. This additional regulatory dialog could help with planning for candidates to skip other common deletions. The early results from our PMO program (with AVI-4658) should help us to streamline this PPMO program; we hope to start a clinical study in mid 2010.

AVI is examining other target deletions with the hope of developing the best candidates for treating these target deletions.

The information set forth above is current only as of the dates noted. While we will make reasonable attempts to keep the information current, there is no guarantee that we will be successful and, except as required under applicable federal and state laws, we disclaim any obligation to do so. Readers are invited to visit the press release and SEC documents sections of this website for more up to date information about the Company and its research, development and clinical programs as well as other aspects of its business.

Information for Physicians, Scientists and Investors

DMD is an incurable muscle–wasting disease caused by mutations in the dystrophin gene. Dystrophin protein plays a key structural role in muscle fiber function by connecting sarcolemma with the intracellular actin filaments. By doing so it prevents damage to the sarcolemma when the cells are force stressed in a functioning muscle. In DMD, dystrophin is missing and the muscle membrane is easily damaged. Although muscle fibers have capacity to regenerate, with age the damage outpaces the repair and the muscle irreversibly deteriorates.

The most frequent mutations in DMD are deletions that disrupt mRNA reading frame. Reading frame disruption occurs when a deleted exon is out of frame, i.e., it does not code for full, three-nucleotide codons and the first and/or last codons are split between adjacent exons, which therefore are not in frame either. Deletions that terminate in in-frame codons, do not prevent translation of dystrophin and result in a truncated protein, still able to bridge actin and sarcolemma. Importantly, these mutations result in the much milder, Becker form of the disease (BMD).

It was found that the precursor to mRNA (pre-mRNA), which carries an out of frame DMD mutation, can be manipulated by sequence specific oligonucleotides designed to skip an out of frame exon during pre-mRNA splicing. The results in DMD patient cells and in animal models demonstrate that such manipulation by splice switching oligonucleotides (SSOs) leads to restoration of the reading frame and production of a truncated but partially functional protein. Thus, SSO drug candidates might offer a treatment for DMD, which is expected to reduce the severity of the muscle disease in a manner analogous to BMD.

The SSO drug candidate AVI–4658 is designed to skip exon 51 of the dystrophin gene, allowing for restoration of the reading frame in the mRNA sequence. Restoration of dystrophin production achieved by skipping this exon might significantly slow the disease process, and could potentially prolong and improve the quality of life for the specifically affected patient.

In principle, approximately 80% of all DMD patients could be treated with exon–skipping drugs. It is important to note, however, that different mutations in the dystrophin gene could require different oligonucleotide drugs. AVI is also evaluating a drug candidate AVI-5038, designed to skip exon 50, which is in preclinical development.

Mutations that could be potentially corrected by skipping exons 51and 50 are as follows:

Exon to be skipped	Drug Candidate	Stage	Treatable Deletions
51	AVI-4658	Clinical Development Phase 1b	45-50, 47-50, 48-50, 49-50, 50, 52
50	AVI-5038	Preclinical Development	51, 51-53, 51-55

References:

Jearawiriyapaisarn et al. Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther. 2008 Sep;16(9):1624–9.
Wu et al, Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci U S A. 2008 Sep 30;105(39):14814–9.
Yin et al Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet. 2008 Dec 15;17(24):3909–18.
Aartsma-Rus et al, Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat. 2009 Jan 20.
Yokota et al. A renaissance for antisense oligonucleotide drugs in neurology: exon skipping breaks new ground. Arch Neurol. 2009 Jan;66(1):32–8.

Importance of RNA splicing

Dystrophin gene and all genes are made of nucleotides and the arrangement or sequence of the four nucleotides A, G, C and T (the letters of genetic code) provides information that determines what protein is made from which gene. In the dystrophin gene, and in most of human genes, this genetic information is not contiguous. Gene fragments that contain the information, called exons, are separated from each other by long-stretches of non-coding fragments, called introns. To make dystrophin protein the DNA from the gene is copied, transcribed, into a pre-mRNA molecule that still contains exons and introns. This pre-mRNA is spliced — that is, the introns are removed and exons are joined together — to make messenger RNA, mRNA, which can be translated into dystrophin protein. In dystrophin, there are 79 exons, most less than 200 nucleotides long. They are separated by much longer introns; the longest one, between exon 44–45, is 248,401 nucleotides long. These introns must be removed and the exons spliced with great precision. If a single nucleotide is missed or an unnecessary nucleotide is left behind, the sequence of mRNA will be incorrect and the dystrophin protein either will not be made or if it is made, it could be faulty.

To make dystrophin protein the genetic information encoded with nucleotides in the gene and the mRNA has to be converted into aminoacids, the building blocks of proteins. The information is translated such that a nucleotide triplet codes for a unique aminoacid. For example, dystrophin exon 49 is 102 nucleotides long and has 34 nucleotide triplets, called codons. This exon is responsible for making a 34 aminoacid long fragment of the dystrophin protein. Exon 50, has 109 nucleotides, i.e., 36 whole codons and one extra nucleotide at the end while exon 51 has 233 nucleotides, 2 extra nucleotides followed 77 full codons. When the adjacent exons 50 and 51 are joined, the extra one and two nucleotides form an additional full codon. When spliced together, these two exons form 114 uninterrupted codons (36+77+1) and serve to make 114-aminoacid piece of dystrophin protein. Like exons 50 and 51, exons 1 and 2, 6–8, 11 and 12, 17–22, 43–46, 50–59 and 60–78 have extra nucleotide and codons that are split between exons. They must be spliced precisely to produce an uninterrupted fragment of dystrophin protein. Exons 3–5,10, 13–16, 23–42,47–49, 60, and 79 contain only full codons.

Gene repair by exon skipping technology

When a dystrophin gene deletion occurs, a gene fragment is missing and two outcomes are possible. For example, deletion of exon 50, which contains one nucleotide of a split codon, will juxtapose exon 49, with full codons, next to exon 51 with 2 extra nucleotides of the split codon. This will reset the codon counter by two nucleotides, forcing it to read A, G, C, T nucleotide letters in a different frame. This will cause wrong aminoacids to be included, making a useless protein or preventing the production of dystrophin altogether. As a result the patient will develop the severe Duchenne form of muscular dystrophy. In contrast, if exon 49 is deleted, full codons will be taken out, and the codon counter continues to read in the same frame, still incorporating the correct aminoacids in the correct sequence. This will produce a truncated form of dystrophin protein which will lacks a small 34-aminoacid internal fragment. A patient with this mutation will develop the milder Becker form of muscular dystrophy.

It was discovered some years ago that inclusion of exon(s) from pre-mRNA into the spliced mRNA can be prevented by blocking a specific exon with a small, synthetic and specific piece of nucleic acid called a Splice Switching Oligonucleotide (SSO). Scientists at AVI have designed SSOs that can enter the muscle cells and skip exons in dystrophin pre-mRNA. AN SSO blocking exon 51 — AVI-4658 — is now being tested in a human clinical trials and an SSO for exon 50 — AVI-5038 — is in animal testing.

The relationship between exon skipping and the DMD deletions is shown below for some of the more frequent deletions in DMD

Exon skipped	Repaired Deletions
51	45-50, 47-50, 48-50, 49-50, 50, 52
50	51, 51-53, 51-55
45	12-44, 18-44, 44, 46-47, 46-48, 46-49, 46-51, 46-53, 46-55
53	10-52, 45-52, 46-52, 47-52, 48-52, 49-52, 50-52, 52
44	10-43, 19-43, 30-43, 35-43, 36-43, 40-43, 42-43, 45, 45-54
8	4-7, 5-7, 6-7, 3-7,
55	47-54, 48-54, 49-54, 50-54, 52-54, 54, 56, 56-62
7	2-6,8-11, 8-17, 8-43, 8-45
52	53, 53-55, 53-57, 53-59, 53-60
17	12-16, 18, 18-20, 18-22, 18-25, 18-27, 18-29, 18-33, 18-36, 18-38, 18-41, 18-44

AVI is currently testing only two candidate drugs, AVI-4658 and AVI-5038, which have been designed to skip exons 51 and 50 respectively.

Clinical Development Status

Clinical Trials

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