Complement Therapeutics and Gene Therapy

3rd Generation Complement Inhibitors

  • Crosstalk:

    • Adaptive immunity implications

    • Intracellular complement considerations

    • Interactions with other systems like coagulation

    • Challenges in delivering therapies across barriers such as the blood-brain barrier (BBB)

  • Generational advancements:

    • 3rd generation: Specific organ delivery

    • 2nd generation: Gene therapy, subcutaneous administration

    • 1st generation: Complement modulation targeting neoepitopes/activation products, longer duration of action

  • Issues with early complement therapeutics:

    • Oral administration challenges

    • Intravenous administration requirements

    • High dosage needs

    • Frequent dosing schedules

    • Total blockade of complement

siRNA Approaches

  • Mechanism Overview:

    • siRNA duplexes are processed by Dicer into single strands.

    • The antisense strand is loaded into the RNA-induced silencing complex (RISC).

    • RISC targets mRNA with complementary sequence, leading to mRNA degradation and silencing.

    • The siRNA/RISC complex is recycled for further silencing.

  • Key Components:

    • RISC: RNA-induced silencing complex

    • Dicer: Enzyme that cleaves dsRNA into siRNA duplexes

    • Ago: Argonaute protein, a component of RISC

    • RdRP: RNA-dependent RNA polymerase, involved in siRNA amplification

  • Cellular Localization:

    • Nucleus: Site of DNA and some RNA processing.

    • Cytoplasm: Location of RISC formation and mRNA degradation.

Targeting the Liver

  • The liver is the main site of complement protein synthesis.

  • siRNA approaches can be used to target C5 RNA in the liver.

  • Delivery methods include subcutaneous administration in mice, rats, and non-human primates (NHP).

Experimental Autoimmune Myasthenia Gravis Model

  • Mice are immunized with 40µg of torpedo acetylcholine receptor (AChR) in Complete Freund's Adjuvant (CFA).

  • Two subcutaneous injections are administered at the base of the tail.

  • ALN-CC5 is used in pre-clinical testing for this model.

Alnylam Pharmaceuticals and Cemdisiran (ALN-CC5)

  • Clinical Studies:

    • Phase 2 study initiated for Atypical Hemolytic-Uremic Syndrome (aHUS).

    • Phase 1/2 study of ALN-CC5 in healthy adult volunteers and patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) - Trial Status: Active, Not Recruiting. ClinicalTrials.gov identifier: NCT02352493

    • Study of Cemdisiran in adults with Immunoglobulin A Nephropathy (IgAN) - Trial Status: Active, Not Recruiting. ClinicalTrials.gov identifier: NCT03841448 ClinicalTrials.gov identifier: NCT03841448

Clinical Trial Data in PNH

  • Dosing Regimens:

    • X = 900mg Eculizumab (Ecu) every 2 weeks with 400mg ALN-CC5 weekly until day 77, then Ecu monthly from day 91.

    • Similar regimen to above, except to day 84, and Ecu from day 98.

    • 1200mg Ecu every 2 weeks to day 56, ALN-CC5 at 200mg to day 77 + Ecu 900mg day 70-140 + Ecu 900 monthly from day 140.

  • Efficacy:

    • 99% inhibition predicted using 600mg ALN-CC5.

    • 5x doses of 400mg effective out to 10 months.

Antisense Technology

  • Mechanism:

    • Antisense strand binds to mRNA, leading to mRNA degradation via RNase H.

  • Chemical Modifications for Antisense Oligonucleotides:

    • 2'-O-methyl (OMe)

    • 2'-O-methoxyethyl (MOE)

    • Locked Nucleic Acid (LNA)

    • Phosphoramidate

    • Peptide Nucleic Acid (PNA)

Targeting Factor B (FB) using ASO

  • Study Design:

    • FB mRNA levels were measured as a percentage of saline control.

    • C3 levels in mg/dL were assessed.

  • Results:

    • FB ASO #1 and FB ASO #2 were tested at 25, 50, and 100 mpk (mg/kg) doses.

    • Significant reduction in FB mRNA levels with both ASOs.

    • C3 levels reduced with FB ASO #2.

Lupus Nephritis Treatment

  • Kidney C3 Deposits:

    • FB ASO #2 reduces C3 deposits in the kidney compared to saline and control ASO.

  • Renal Pathology Score:

    • MRL/LPR model shows improved renal pathology scores with FB ASO #2.

  • Clinical Outcomes:

    • Reduced proteinuria incidence in NZB/W f1 model.

    • Improved survival rates with FB ASO #1 and FB ASO #2.

Knockdown of C3 in Kidney Disease

  • C3 siRNA tracking in C57Bl/6 mice using IVIS (in vivo imaging system).

  • Time points: 4hr, 24hr, 48hr, 72hr, 96hr, 7d, 10d, 14d, ex vivo.

  • Various siRNAs tested (Mir-1.00, Mo-181e, etc.).

Personalized Medicine - Batten's Disease Treatment

  • Description: Treatment of one patient with Batten's Disease (a rare and fatal neurodegenerative disease) with milasen.

  • Study Timeline:

    • Referral, establishment of patient cell lines, identification of mutation and splice defect.

    • Identification of ASO hit and validation of ASO functionality.

    • Permission to proceed, start of toxicologic study in rats, and first patient dosing.

  • Genetic Diagnosis:

    • Identification of SVA insertion in MFSD8 gene.

  • Effects of SVA Insertion:

    • Abnormal MFSD8 splicing and translation.

N=1 Clinical Study - Milasen Dosing

  • Dosing Schedule:

    • Escalating loading doses approximately every 2 weeks.

    • Maintenance doses approximately every 3 months.

  • Milasen in CSF:

    • Milasen concentration in CSF before each injection.

  • Seizure Trends:

    • Seizure trends as reported by parents and detected by EEG.

Age-Related Macular Degeneration (AMD)

  • Description:

    • AMD causes irreversible sight loss and is the leading cause of blindness in the western world.

    • Threatens 1-in-5 people over the age of 65 (WHO).

  • Pathology:

    • Begins with drusen, which are focal depositions of acellular debris between the retinal pigment epithelium and Bruch’s membrane.

Complement in AMD

  • Evidence:

    • Complement components identified in Drusen.

  • Genetic Association:

    • Genome-wide association studies (GWAS) have linked complement components and AMD.

    • Related factors: complement factors H (CFH), I (CFI) and B (CFB) in addition to the complement components 2 (C2) and 3 (C3)

    • The SNP rs1061170 (Y402) in hCFH is a key susceptibility factor.

    • 30% of Europeans have FH-Y402H on one allele, increasing AMD risk.

  • Therapeutic Strategy:

    • Intervention in the human complement system is a promising strategy for AMD treatment.

Pre-Clinical Modeling of AMD

  • Models:

    • Wet AMD – CNV (choroidal neovascularization) – fairly easy to model.

    • Dry AMD with Geographic atrophy – challenging.

  • Limitations:

    • Mice do not have a macula.

  • Capabilities:

    • Can model early AMD.

Smoking Mice - Model of AMD

  • Smoking chamber setup for mice to model AMD.

AMD Phenotype – Smoking Mice vs. Man

  • Comparison of AMD phenotypes in humans and smoking mice.

  • Markers Used

    • CD63

    • αB crystallin

AAV in Action

  • Reference: Li, C., and Samulski, R.J. (2020). Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 21, 255-272.

TT30 (CR2-FH)

  • Targeting C3d with CR2-FH fusion protein for complement regulation.

AAV5-Mediated Expression of CR2-fH in the Mouse RPE

  • Reference: Mol Ther Methods Clin Dev. 2017 Nov 10;9:1-11. doi: 10.1016/j.omtm.2017.11.003.

AAV5 CR2-FH Protects Against Smoke-Induced Changes

  • Experimental Groups:

    • Room air

    • mCherry

    • CR2-fH

    • Smoke exposure

  • Results:

    • CR2-fH reduces the percentage of thickened Bruch’s membrane (BrM) in smoke-exposed mice.

Subretinal CR2-FH Expression

  • Optimal Expression Site:

    • Subretinal injections are optimal for expression in the RPE/choroid.

  • Delivery Methods:

    • Subretinal injections: Target RPE/choroid.

    • Intravitreal injections: Target retina.

  • Transport Across RPE:

    • CR2-fH transport from apical to basal side.

Gene Therapy - Complement Factor I in the Eye

  • AAV Vector Used:

    • AAV-CAG-CFI or CFI.co-WPRE-bGHPA

  • Experimental Groups:

    • Sham

    • AAV.CFI High dose

  • Visualization:

    • Phalloidin staining

    • 3D view

Gyroscope - Vision for Life (Novartis Company)

  • FOCUS [NCT03846193]: Phase I/II trial for GA secondary to AMD, evaluating GT005 with subretinal injection, enrolling up to 60 participants with 48 weeks follow-up.

  • HORIZON [NCT04566445]: Randomized Phase II trial for GA secondary to AMD, evaluating two doses of GT005 with single subretinal injection, enrolling up to 180 participants with 48 weeks follow-up.

  • EXPLORE [NCT04437368]: Randomized Phase II trial for GA secondary to AMD with rare CFI variants, evaluating two doses of GT005 with single subretinal injection, enrolling up to 75 participants with 48 weeks follow-up.

One-Time Gene Therapy May Offer Durable Therapeutic Effect

  • Gene therapy is designed to provide a durable effect with a single administration, unlike IVT therapies that require repeat injections to maintain effect.

FOCUS Trial - GT005 Safety and Dose Response

  • Phase I/II open-label trial with subretinal transvitreal delivery.

  • Dose Escalation (complete).

  • Dose Expansion (complete).

Orbit SDS Delivery System

  • Used for subretinal microinjection. Indicated for microinjection into the subretinal space with Balanced Salt Solution (BSS).

FOCUS Trial - GT005 Tolerability

  • GT005 has been well tolerated in cohorts 1-4 (March 2022 Safety Analysis, N=31).

  • No GT005-related Serious Adverse Events (SAEs).

  • No safety signal on laboratory parameters.

  • RPE changes noted in some subjects in the high-dose group, restricted to the bleb area without significant visual changes.

FOCUS Trial - Interim Data on Inflammation

  • No clinically significant GT005-related inflammation observed.

  • GT005 is associated with clinically benign immunogenicity.

  • No antibody-mediated immunogenicity to the CFI transgene.

  • Vector shedding profile aligns with other rAAV ocular therapies.

  • AAV2 neutralizing antibodies (n=20): Pre-existing antibodies in 11 patients, transient increase in 1 patient.

  • T-cell immunogenicity (n=25): Pre-existing T-cell immunogenicity in 1 patient, low magnitude increase in 2 patients.

  • Anti-FI antibodies (n=20): No anti-FI antibodies detected to date.

  • AAV2 vector shedding (n=21): No vector detected in urine or blood, low levels in saliva or tears of 5 patients.

Vitreous FI and Downstream Proteins

  • GT005 generated sustained increases in vitreous FI and decreases in downstream proteins involved in overactivation of the complement system.

  • Increase in Vitreous
    FIFI

  • Decrease Vitreous
    BaBa
    C3C3
    C3b/iC3bC3b/iC3b

EXPLORE Trial Discontinuation

  • Phase II clinical trial studying GT005 in geographic atrophy (GA) secondary to AMD with rare CFI variants.

  • Trial discontinued based on recommendation from the independent Data Monitoring Committee due to futility criteria being met.

Immune Response to AAV

  • Triggers:

    • AAV serotype

    • Total vector dose

    • PAMPs (capsid, Me CpG, dsRNA)

    • DAMPS (tissue damage)

    • Route of administration

  • Outcomes:

    • Loss of transgene expression

    • Re-administration blocked

    • Complement activation

    • Cytopenias?

Complement Activation and Immune Response to AAV

  • Complement activation is integral to the immune response to AAV.

  • Measured by neutralizing antibody titer.

C Inhibitors to Protect AAV Therapy

  • The use of C inhibitors to protect AAV therapy may be beneficial.

  • Structure formula of APL-9

Targeting Complement in the Brain & BBB

  • Parkinson's Disease: CR3 knockout mice were protected from dopaminergic neuron loss and motor dysfunction.

  • Huntington's Disease: Oral administration of C5aR antagonist reduced weight loss and motor deficits in an animal model.

  • Amyotrophic Lateral Sclerosis: Oral administration of PMX205 reduced weight loss and motor deficit scores and slowed disease progression.

  • Alzheimer's Disease: PMX205 decreased amyloid and tau deposits, reduced activated glia, and improved cognition.

Anti-C7 Monoclonal Therapy

  • Anti-C7 is highly effective in experimental autoimmune myasthenia gravis (EAMG) model.

Getting Drugs/Anti-C7 mAb Across the BBB

  • Mechanisms for drugs to cross the blood-brain barrier (BBB).

Final Thoughts

  • Gene therapy is a promising field, particularly for targeting complement proteins.

  • There are many challenges to AAV-based therapy and the ability to control complement activation on AAV may be a great help.

  • Complement gene replacement therapy in the eye is showing promise and avoids the issues of systemic AAV delivery.

  • Complement in brain disease provides new challenges and the need for bespoke therapies with unique modality.

  • The future will be highly targeted local complement regulation.