Chemically Modified Nucleic Acids as Therapeutics PDF

# Chemically Modified Nucleic Acids as Therapeutics ## Oligonucleotides That Affect Splicing of Pre-mRNA - Muscular Dystrophy (Sarepta) - Spinal Muscular Atrophy (Ionis and Biogen) ## shRNAs That Trigger Degradation of Target mRNAs - Transthyretin amyloidosis (Alnylam) ## Chemically Modified DNA...

# Chemically Modified Nucleic Acids as Therapeutics ## Oligonucleotides That Affect Splicing of Pre-mRNA - Muscular Dystrophy (Sarepta) - Spinal Muscular Atrophy (Ionis and Biogen) ## shRNAs That Trigger Degradation of Target mRNAs - Transthyretin amyloidosis (Alnylam) ## Chemically Modified DNA and RNA as Therapeutics ### Phosphorothioate (PS) Backbones - Phosphorothioate (PS) backbones, as well as 2'-O-methoxyethyl (2'-MOE) and 2'-O-methyl (2'-OMe) substituents, increase resistance to degradation and promote protein binding to target RNA. ### 2'-OMe - Prevents cleavage of the oligonucleotide. ## Chemically Modified DNA and RNA as Therapeutics ### Phosphorodiamidate Morpholino Oligomers (PMOs) - In phosphorodiamidate morpholino oligomers (PMOs), ribose (RNA) or deoxyribose (DNA) is replaced with morpholine rings, and the phosphorothioate or phosphodiester (RNA) groups are replacing with uncharged phosphorodiamidate groups, resulting in a compound that is neutral, taken up by cells, and very resistant to degradation. ## Most Precursors of Human Messenger RNAs Undergo RNA Splicing in the Nucleus to Remove Intervening Sequences (Introns) - The nucleus contains rRNA, tRNA, and mRNA. - mRNA is translated into protein. ## Splicing Involves Many RNA-Protein Complexes and Several Well-Understood Biochemical Reactions - Exon 1 is separated by 5' SS and GU. - Branch point is marked by A. - Exon 2 is separated by 3' SS and AG. - Cleavage at 5’ splice site then forms a lariat-like intermediate. - Cleavage at 3’ splice site then ligates the exons. ## Oligonucleotides That Affect Splicing of Pre-mRNA - Muscular Dystrophy (Sarepta) ## Duchenne's Muscular Dystrophy (DMD) - Most common type of muscular dystrophies. - Affects 1 in 3300 boys. - X-linked recessive disorder. - Mutation of dystrophin gene. - Gower's maneuver. - Boys affected by DMD are usually diagnosed at 3-5 years of age. - Patients go into a wheelchair at about 12 years of age. - Patients usually die in their 20s from respiratory or heart failure. - A minority of patients show mental impairment ## The Structure of a Multinuclated Striated Muscle Cell - The sarcolemma is the cell membrane. - T-tubules form a network throughout the muscle cell. - The sarcoplasmic reticulum stores calcium. - The myofibrils are composed of: - thin filaments - thick filaments - The sarcoplasm contains mitochondria. - One sarcomere is compose of: - H-band - A-band - M-line - I-band - Z-line ## Dystrophin - the Largest Human Protein - Dystrophin has a long central rod domain made up of 24 spring-like “spectrin repeat” linkers, which are interspersed with “hinge” regions. Each spectrin repeat is encoded by a single exon. - Dystrophin binds to the dystrophin-associated protein complex through its essential C terminus, and the N terminus of dystrophin binds to the actin cytoskeleton. Dystrophin provides a strong, flexible, mechanical link between the intracellular cytoskeleton and the extracellular matrix. - Loss of integrity of the cell surface membrane occurs when a mutant dystrophin (or other protein of the DAPC) is expressed, resulting in muscle fibers that are more susceptible to damage. ## Becker's Muscular Dystrophy - In frame exon deletions of one or more exons encoding one or more spectrin repeats lead to shorter, but still reasonably functional, dystrophin proteins. - The important N-terminal domains that bind to the actin cytoskeleton and the C-terminal domains that bind to cell surface membrane proteins are intact and functional. - These properties of the mutant dystrophin in Becker’s Muscular Dystrophy suggest that simply deleting (or blocking the RNA splicing of) an exon or exons encoding one or more spectrin repeats would generate a mutant dystrophin with fewer than normal numbers of spectrin repeats but still be reasonably functional. ## Molecular Approaches Towards a Therapy for Duchenne's Muscular Dystrophy - Splicing involves binding of several small RNA protein complexes to specific sequences (splice donor site, splice acceptor site, splicing enhancer and splicing inhibitory) sites in the pre-mRNA. - Binding of a synthetic Antisense Oligonucleotide to the nuclear pre-mRNA blocks the binding of one or more of these factors and prevents splicing of that exon. ## Phosphorodiamidate Morpholino Oligomers (PMOs) - Uncharged backbone. - Resistant to degradation. - Base pair binding to RNA; can inhibit RNA splicing. ## Exon Skipping: Exondys 51 - Mutant exons that result in out-of-frame translation may be deleted by skipping the exon. - **Exondys 51:** Uses a PMO to skip exon 51 in dystrophin to restore the reading frame and produce a shorter but functional dystrophin. ## Exondys 51 Trial Results - Phase I/II Trial (2016): 0.93% of normal dystrophin levels restored by drug. - Study 203 (2016) - Phase III Trial: ongoing. - FDA accelerated approval for commercial use. - Not approved in Europe. - Effect size, Placebo? ## Spinal Muscular Atrophy (SMA) - Autosomal recessive. - Loss of both copies of the SMN1 gene. - Death of a-motor neurons in spinal cord. - Symptoms: muscle weakness, atrophy of voluntary muscles, paralysis, infantile death. - Affects 1 in 6,000 live births. - Until recently no treatment available! ## Antisense Oligonucleotides to Enhance Correct Splicing of the Nuclear SMN2 Pre-mRNA - The SMN1 and SMN2 genes encode the same protein. Spinal muscular atrophy is caused by autosomal recessive (both copies mutant) of the SMN1 gene. - The severity of the disease depends on how much functional SMN protein is made from the SMN2 gene - but it is usually not enough to prevent lethality at an early age. - The reason is that the SMN2 gene has a slightly different DNA sequence that causes Exon 7 not to be spliced in to the messenger RNA, resulting in production of a mutant SMN protein that lacks essential sequences and is nonfunctional. ## Nucleic Acid Therapy for Spinal Muscular Atrophy Type I Schematic Representation of the Mechanism of Action of Nusinersen (Biogen's Spinraza) - SMN2 Inron 7 contain an intronic splicing silencer with a binding site for negative splicing factors.. - Binding of these negative splicing factors to intron 7 pre-mRNA prevents splicing of exon 7. ## Nucleic Acid Therapy for Spinal Muscular Atrophy Type I Schematic Representation of the Mechanism of Action of Nusinersen (Biogen's Spinraza) - The antisense oligonucleotide nusinersen binds to and blocks the ISS-N1 site, preventing the binding of the negative splicing factors, and allowing splicing to proceed. - As a result, Exon 7 is included in the SMN2 mature RNA, and the mRNA is translated into full-length SMN protein. ## Splicing Correcting Therapy for Spinal Muscular Atrophy ### Spinal Muscular Atrophy Frequency - A common orphan disease: - 1 in 10,000, born with spinal muscular atrophy. - 1 in 50, is a carrier. - Patients worldwide: - North America: 14,000 - Europe: 10,000 - Japan: 1,000 ### Types of SMA - Type I: Severe - Life expectancy <2 years, never sits independently - Type II: Intermediate - Life expectancy >2 years, sits but never walks independently - Type III: Mild - Able to stand and walk limited steps. ### Timeline for Treatment - 1995: Discovery of SMN1 deletions as cause of SMA. - 1996-1997: Discovery of SMN-Gemins complex and its function in snRNP/spliceosome biogenesis. - 1997: SMN protein level and snRNP assembly correlate with SMA severity. - 1999: SMN2 single-nucleotide change causes exon 7 skipping. - 2000: SMN increase alleviates SMA phenotypes in mouse models. - 2006: Identification of a targeting sequence in SMN2's intron 7 for ASO-mediated correction of exon 7 skipping. - 2008-2011: ASO improves exon 7 inclusion, SMA symptoms in mice. - 2011: ASO improves exon 7 inclusion, SMA symptoms in mice. - 2014: Phase III clinical trials (IONIS-SMN/nusinersen). - 2016: FDA approval of Spinraza. ## shRNAs That Trigger Degradation of Target mRNAs - Transthyretin amyloidosis (Alnylam) ## What is Transthyretin? - Transports the hormone thyroxine in the blood. - Not necessary for normal health. - A 55kD tetramer of identical subunits. - Association of two of dimers in a face-to-face fashion produces the homo-tetrameric structure and creates the two thyroxine binding sites per tetramer. ## Transthyretin Amyloidosis - Autosomal dominant neurodegenerative disease (only one copy of the defective gene is sufficient to cause the disorder). - Most commonly a mutation of valine to methionine at amino acid 30. - Mutation causes dissociation of tetramers into miss-folded monomers that aggregate into a variety of structures including amyloid fibrils. - Because most patients are heterozygotes, they deposit both mutant and wild type transthyretin subunits. ## Hereditary ATTR (hATTR) Amyloidosis Patisiran and ALN-TTRsc02 - Description: - Mutations in TTR gene lead to deposition of miss-folded protein as amyloid, causing multi-system disease manifestations. - Significant morbidity and fatal within 2-15 years from symptom onset. - Patient Population* ~50,000 worldwide. - Cardiac: Heart failure, arrhythmia. - GI: Diarrhea, nausea, vomiting. - GU: Proteinuria, kidneyfailure, UTI, incontinence, impotence. - Autonomic: Falls, lightheadedness, weightloss. - Peripheral: Numbness/tingling, pain, weakness, impaired walking. ## Patisiran - Alnylam's FDA-Approved Drug to Treat Transthyretin Amyloidosis - Double-stranded small interfering ribonucleic acid (siRNA), formulated in lipid nanoparticles for delivery to hepatocytes. - Specifically binds to a genetically conserved sequence in the 3'-untranslated region (3'-UTR) of both mutant and wild-type transthyretin (TTR) messenger RNA (mRNA), triggering degradation of the TTR mRNA. - All of the pyrimidines in the sense strand (top) and two of the uridines in the antisense strand (bottom) contain 2'-O-methyl modified ribonucleotides. - All internucleotide linkages are natural, chemically unmodified phosphodiester linkages. ## MicroRNA Biogenesis and Function in Animal Cells - MicroRNAs are 21-23 nucleotide long RNAs that bind to target messenger RNAs and either block their translation into protein or trigger their cleavage. - Humans produce over 3000 different microRNAs. ## microRNA and shRNA Biogenesis - miRNA Gene: - Transcription, splicing, polyadenylation. - Pri-miRNA. - Drosha, Pasha. - Pre-miRNA. - Exportin 5. - Pre-miRNA. - Dicer/loquacious. - miRNA. - miRNA: - Guide strand. - RISC. - Passenger strand. - mRNA: - Cleavage and degradation. - P body. - RNA degradation. - Target mRNA. ## Two Modes of RNA Interference - MicroRNA: - Processed from intracellular miRNA precursor. - Incomplete base pairing with mRNA - thus less selective with more targets. - (miRNA) - Short helical interfering RNA (shRNA): - Processed from introduced double-stranded (ds) RNA. - More exact base matching – fewer and more specific mRNA targets. - Leads to degradation of specific target mRNA. ## RNA Interference Using Short Helical Interfering RNAs (shRNAs) - dsRNA - shRNA - siRNA duplex - Formation of RISC. - siRNA/mRNA-complex. - Sliced mRNA. ## shRNAs That Trigger Degradation of Target mRNAs - Transthyretin amyloidosis (Alnylam). ## Patisiran - Alnylam's FDA-Approved Drug to Treat Transthyretin Amyloidosis - This phase 3 trial randomly assigned patients with hereditary transthyretin amyloidosis with polyneuropathy, in a 2:1 ratio, to receive intravenous patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks. - The primary end point was the change from baseline in the modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment) at 18 months. - Other assessments included the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) questionnaire (range, -4 to 136, with higher scores indicating worse quality of life), 10-m walk test (with gait speed measured in meters per second), and modified body-mass index (modified BMI, defined as [weight in kilograms divided by square of height in meters] × albumin level in grams per liter; lower values indicated worse nutritional status). - A total of 225 patients underwent randomization (148 to the patisiran group and 77 to the placebo group). - The mean (±SD) mNIS+7 at baseline was 80.9±41.5 in the patisiran group and 74.6±37.0 in the placebo group; the least-squares mean (±SE) change from baseline was -6.0±1.7 versus 28.0±2.6 (difference, -34.0 points; P<0.001) at 18 months. - The mean (±SD) baseline Norfolk QOL-DN score was 59.6±28.2 in the patisiran group and 55.5±24.3 in the placebo group; the least-squares mean (±SE) change from baseline was -6.7±1.8 versus 14.4±2.7 (difference, -21.1 points; P<0.001) at 18 months. - Patisiran also showed an effect on gait speed and modified BMI. At 18 months, the least-squares mean change from baseline in gait speed was 0.08±0.02 m per second with patisiran versus -0.24±0.04 m per second with placebo (difference, 0.31 m per second; P<0.001), and the least-squares mean change from baseline in the modified BMI was -3.7±9.6 versus -119.4±14.5 (difference, 115.7; P<0.001). - Approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups. - Conclusions: - In this trial, patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO Clinical Trials .gov number, NCT01960348.)