RNA-based therapeutics for the treatment of blood disorders: A review and an overview

SiRNA therapy

RNA interference (RNAi), also referred to as post-transcriptional gene silencing, is an evolutionarily conserved process that lowers gene expression by targeting and degrading specific mRNA molecules with RNA molecules. In this RNAi pathway, siRNA plays a crucial role. Gene expression is suppressed when short RNA molecules, such as microRNAs (miRNAs) or siRNAs, bind to and deactivate endogenous mRNA. The Nobel Prize in Physiology or Medicine 2006 was awarded jointly to Boyce S, Rangarajan S for their discovery of RNAi.^[5]

RNA therapy in hemophilia

Unmet needs and challenges with current treatment options^[8]

1. Limited protection from current prophylaxis: Regular FVIII replacement therapy, administered 2–3 times a week, provides inconsistent protection due to the short half-life of the infused FVIII.

2. High frequency of anti-FVIII antibodies: Approximately 25–30% of patients with severe hemophilia A and 3–13% with moderate or mild hemophilia develop anti-FVIII antibodies. More than 95% of these inhibitors develop within the first 50 days of treatment.^[9]

3. Breakthrough bleeding despite prophylaxis: Patients with FVIII inhibitors are still at risk for significant and potentially life-threatening bleeds, even when receiving the recommended doses of on-demand or prophylactic bypassing agents.

4. Difficulties with venous access: All current hemophilia treatments involving clotting factor concentrates (CFCs) require intravenous infusion, which poses a challenge for patients.

5. Rigorous immune tolerance induction (ITI) regimen: ITI therapy requires high doses of CFCs and frequent infusions every 1–2 days, potentially lasting 18– 24 months, with success rates of about 60–70% in individuals with hemophilia.

Hence, eliminating the inhibitors of coagulation by employing non-factor replacement therapies that either block or remove anticoagulants has demonstrated promise in restoring hemostasis in hemophilia without the need to replace the missing clotting factor.

Fitusiran:^[5] It is a siRNA-based treatment developed to prevent bleeding in hemophilia A and B. This synthetic siRNA is chemically attached to a triantennary N-acetylgalactosamine (GalNAc) ligand. Th GalNAc-siRNA complex specifically targets and reduces the mRNA of antithrombin, leading to a decrease in antithrombin production. As a result, there is enhanced inhibition of thrombin and other serine proteases, including factors Xa, IXa, and XIIa.

In the phase 1 dose escalation study by Pasi et al.,^[10] the safety and tolerability of subcutaneous fitusiran were assessed in four healthy volunteers and 25 adults with moderate to severe hemophilia A and B who did not have inhibitors. The healthy volunteers received a single subcutaneous dose of fitusiran at 0.03 mg/kg of body weight. The hemophilia participants received three doses of fitusiran, either weekly (at doses of 0.015, 0.045, or 0.075 mg/kg) or monthly (at doses of 0.225, 0.45, 0.9, or 1.8 mg/kg, or a fixed dose of 80 mg).

Peak plasma levels of fitusiran were observed 2–6 h after administration. In patients with hemophilia, plasma levels of fitusiran rose while antithrombin levels decreased in a dose-dependent manner. The reduction in antithrombin was associated with increased thrombin generation, with similar outcomes in both hemophilia A and B patients. The pharmacological effect was prolonged, with antithrombin recovery occurring at a median rate of 10–15% per month after discontinuing fitusiran.

Fitusiran was generally well tolerated, with injection site reactions and arthralgia being the most common side effects. Transient increases in liver aminotransferases were observed in 36% of participants, but no patients developed anti-fitusiran antibodies. Monthly fitusiran dosing was well tolerated, lowered antithrombin levels from baseline, and improved thrombin generation. It was concluded that monthly treatment with fitusiran could help reduce bleeding episodes. Ongoing phase III trials for fitusiran are ATLAS-PEDS, ATLAS-OLE, ATLAS-PPX, ATLAS-INH, and ATLAS A/B.^[11] Other potential targets for RNAi therapy are protein S and heparin cofactor II.^[10]

RNA therapy in thalassemia

In β-thalassemia, the condition arises from a single point mutation or a small deletion in the β-globin gene. The primary goal of treatment is to achieve a balanced ratio of α to β chains, which helps minimize the surplus of free α-chains.^[12]

Several nucleic acid-based approaches could serve as precision medicine options for β-thalassemia, including:

1. Decreasing α-globin expression-^[13] To achieve a balanced α/β-globin ratio, it is essential to reduce α-globin mRNA expression by 25–50%, while maintaining stable levels of β-globin mRNA. siRNA or miRNA form complexes with the RISC, which then cleaves the mRNA with the help of RNAse, resulting in reduced α-globin expression.

2. Modifying β-globin pre-mRNA splicing to correct mutations-^[14] A major molecular mechanism behind the defect in β-globin gene expression is mutation-induced abnormal splicing (e.g., mutations such as IVS1–5, IVS1–6, and IVS1–110 in intron 1), which activates non-canonical splice sites and disrupts normal splicing pathways. Such splicing errors also lead to the formation of abnormal hemoglobins, like HbE. The activation of these atypical splice sites causes incorrect RNA splicing, resulting in the retention of intron fragments and the production of non-functional β-globin chains. Short synthetic oligonucleotides (15–25 nucleotides) targeting specific pre-mRNA sequences can alter the recognition of splice sites by the spliceosome. This modification influences the splicing of the targeted transcript, which is why these oligonucleotides are called splice-switching oligonucleotides (SSOs). Mechanisms by which SSOs modulate RNA splicing: Exon skipping, Exon retention and Restoration of correct splicing.

3. Reactivating γ-globin expression-^[15] Elevated levels of HbF can help reduce the clinical severity of β-thalassemia, especially in cases co-inherited with HPFH or the Xmnl1-HBG2 polymorphism. The increased γ-globin chains interact with surplus α-globin chains, improving the balance between the globin chains and mitigating the disease’s severity. The expression of the γ-globin gene is controlled by three transcription factors: BCL11A, KLF1, and MYB, which suppress its expression after birth. siRNA can be utilized to inhibit BCL11A production, effectively boosting HbF levels.

RNA therapy in porphyria

Acute hepatic porphyria refers to a collection of inherited metabolic disorders that impair heme production in the liver. Enzyme deficiencies cause a buildup of neurotoxic or phototoxic heme precursors, resulting in symptoms associated with neurovisceral problems or sensitivity to light.^[16]

Givosiran

It is an siRNA targeting 5-aminolevulinic acid synthase (ALAS1), which is covalently linked to a ligand containing three GalNAc residues to enhance delivery to hepatocytes.^[17] The aim is to reduce ALAS1 activity and lower the levels of toxic heme intermediates, primarily 5-aminolevulinic acid and porphobilinogen.

In the phase III trial by Balwani et al.,^[18] 94 patients with acute hepatic porphyria were randomly assigned to receive either subcutaneous givosiran (2.5 mg/kg body weight) or a placebo once a month for 6 months. The primary endpoint was the annualized rate of composite porphyria attacks in patients with acute intermittent porphyria, the most common form of acute hepatic porphyria.

The mean annualized attack rate was 3.2 in the givosiran group, compared to 12.5 in the placebo group, demonstrating a 74% reduction in attacks in the givosiran group (P < 0.001). In patients with acute intermittent porphyria, givosiran led to lower urinary levels of ALA and porphobilinogen, fewer days requiring hemin treatment, and improved daily pain scores compared to the placebo.

Common adverse events in the givosiran group included elevated serum aminotransferase levels, changes in serum creatinine levels and estimated glomerular filtration rate, and injection-site reactions.

Gene-based therapies currently under development show considerable promise for improving patient care. New treatments being explored, such as pharmacological chaperones, aim to stabilize or activate wild-type hydroxymethylbilane synthase and its mutants associated with acute intermittent porphyria. These therapies could extend the enzyme’s half-life, improve its function, and potentially offer preventative treatment for the disease.

RNA therapy in amyloidosis

Transthyretin (TTR) amyloidosis occurs when the TTR protein misfolds. TTR is a tetramer produced by the liver and functions as a transport protein for thyroxine and retinol. There are two types of TTR amyloidosis.^[19]

1. Wild-type ATTR, which occurs secondary to an acquired pathogenic process associated with aging.

2. Hereditary ATTR (hATTR or ATTRv), which occurs secondary to an inherited TTR-gene mutation.

   • SiRNA therapy-Patisiran was the first siRNA developed and received approval from the United States Food and Drug Administration (FDA) and the European Commission in 2018 for treating ATTRv-PN. It is delivered as a lipid nanoparticle, which separates the double-stranded siRNA into single strands. Once released into the hepatocyte cytoplasm, the single-stranded siRNA binds to complementary mRNA, activating the Ago slicer protein. This results in the degradation of the mRNA and inhibits TTR synthesis.^[20]

In the APOLLO phase 3 trial,^[21] done on 225 patients, patisiran (at a dose of 0.3 mg/kg for those under 100 kg and 30 mg for those over 100 kg) demonstrated a significant improvement from baseline in the modified Neuropathy Impairment Score + 7 in the treatment group compared to the placebo group. In addition, it showed improvements in the Norfolk Quality of Life Questionnaire-Diabetic Neuropathy score and gait speed.

Other siRNA therapy drugs in development include revusiran and vutrisiran.^[22,23]

ASO- Inotersen: It is a 2’-O-methoxyethyl-modified ASO that targets the 3’ untranslated region of complementary mRNA, leading to its degradation through ribonuclease H1-mediated action. It was the first ASO to be developed and approved by the FDA and EMA in 2018 for treating ATTRv-PN.^[24]

In the phase III NEURO-TTR^[24] trial, in adults with stage 1 (ambulatory) or stage 2 (ambulatory with assistance), hereditary TTR amyloidosis with polyneuropathy was randomly assigned in a 2:1 ratio to receive weekly subcutaneous injections of inotersen (300 mg) or placebo. The study concluded that inotersen improved the progression of neurological disease and quality of life in patients with hereditary TTR amyloidosis. Thrombocytopenia and glomerulonephritis were managed through increased monitoring.^[25]

Eplontersen is another ASO for the treatment of hTTR amyloidosis-related polyneuropathy, with ongoing evaluation.^[26]

Future directions

Potential of RNA therapy lies in its ability to precisely target and regulate the expression of genes involved in the development and progression of these cancers. It works by gene silencing, modulating gene expression, overcoming drug resistance, and targeting immune checkpoints and leukemic stem cells along with providing personalized treatment. As shown in Table 2 below, RNA therapeutics are being evaluated in a number of other hematological disorders including acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndromes (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM).^[1] Table 3 shows FDA approved RNA therapeutics in other disorders.^[2]

Advantages of RNA therapy

• Targeting the untargetable, treating the untreatable

• Quick production

• Patient customized therapy.

Disadvantages of RNA therapy

• Poor bioavailability

• Short lived response

• Off target effects.

Status of RNA therapeutics in India

Although RNA therapeutics are gaining considerable global attention, their development in India is still in the early phases, with challenges such as a lack of effective delivery systems, limited research on silencing agents, and difficulties in translating nucleic acid-based therapies into clinical trials. However, there is increasing interest and efforts to build domestic capabilities in this field, including collaborations with international research institutions to address these challenges and advance RNA therapeutics in India.^[27]