Surgical delivery of PPY988 AAV2-CFI.

(A) PPY988 vector design. FI protein is secreted by cells targeted by AAV2 serotype and containing the cDNA sequence for wild-type CFI (NM_000204.4) driven by a CAG promoter 8. (B) Subretinal delivery of PPY988. (C) Vitrectomy during SR delivery of PPY988 results in loss of total protein (TP) in the vitreous humor. TP concentration (y-axis, log2 µg/µL) was measured at baseline and 5 or 12, 36, and 96 weeks post-surgery (y-axis). Mean +/- SE is shown by blue bars and lines whilst all subjects are shown by black lines for both vitreous (left) and aqueous (right) humor. A significant reduction in TP was observed at week 96 in vitreous humor (*P=0.033). Significance is defined as *P<0.05. BL = Baseline; WK= week.

Temporal expression of factor I (FI) in vitreous humor (VH) and aqueous humor (AH) after PPY988 delivery.

(A,B) FI levels (y-axis, ng/mL) in VH were significantly increased at week 36 and 96 post-surgery (x-axis) both when non-normalised (A) (P=0.001 for both timepoints) and total protein-normalised (B) (P=0.002 at both timepoints). Mean +/- SE is shown by bars for all (black lines) broad (blue lines) and CFI RV (green lines) groups. (C,D) FI level (y-axis, ng/mL) for each subject in the cohort is tracked by week (x-axis) and separated by dose (2E10 vg: orange lines; 5E10 vg: green lines; 2E11 vg: blue lines) in VH (C) and AH (with significant increase in AH FI at week 36 and 96 (both P<0.001)) (D), where mean +/- SE shown by black bars. Significance is defined as *P<0.05; **P<0.01, ***P<0.001. N numbers are shown above X-axis. BL = Baseline; WK= week.

Modulation of Ba and Ba:FB by PPY988 delivery.

(A,B) Ba concentrations (Y-axis, ng/mL) in vitreous humor (VH) samples by week post-surgery (X-axis). Mean +/- SE is shown by bars for all (black lines) broad (blue lines) and CFI RV (green lines) groups. Significant reductions in Ba were observed at week 36 for both non-normalised (P=0.004) (A) and total protein (TP) normalised (P=002) (B) data and week 96 for non-normalised data (P=0.003) and TP normalised (P=038). (C,D) Ratios of Ba:FB concentration (y-axis, ng/mL) were calculated for each subject at each sampling post-surgery week (x-axis). Mean +/- SE is shown by bars for all (black lines) broad (blue lines) and CFI RV (green lines) groups. Significant reductions were identified at week 36 for both non-normalised (P<0.001) (C) and TP normalised (P<0.001) (D) Ba:FB ratios, with a reduction at week 96 for non-normalized (P=0.001) and TP normalized (P=0.033) ratios. Significance is defined as *P<0.05; **P<0.01, ***P<0.001. N numbers are shown above X-axis. BL = Baseline; WK= week.

Modulation of complement AP and CP biomarkers by PPY988 delivery.

Fold-changes (y-axis) in complement biomarker [FI, C3, FH, FB, C1q, Ba, C3a, C3b/iC3b, C4b, Ba:FB, C3a:C3, C3b/iC3b:C3] concentrations from baseline at each post-surgery week (VH: week 5/12, 36 and 96; AH: week 36 and 96; X-axis) are shown in VH with no normalisation (A), VH with total protein normalisation (B), and AH with no normalisation. Mean is shown by thick black lines, +/- SE is shown by bars. Individual subject changes are shown by grey lines. Yellow boxes highlight disparate trends in VH vs AH biomarkers. BL = Baseline; WK= week.

In vitro modelling of vitreous humor complement activation inhibition by FI vs pegcetacoplan and additional complement regulators.

FI (orange), FH (purple), and pegcetacoplan (blue) were titrated (x-axis, ng/mL) through artificial vitreous complement matrix on LPS-coated WeislabTM plates and C3a generation was measured by ELISA (y-axis, ng/mL). C3a is a marker of convertase activity in the complement activation cascade, the potency of complement inhibitors or regulators is reflected in lower amount of C3a generated in the assay when testing comparable concentrations. Mean is shown with +/-SD represented by bars and 4-parameter fit logistic regression curves are shown by lines. The 1st-3rd quartile of increased FI expression from baseline after 36 weeks of PPY988 delivery is shown by orange area. The calculated trough concentration of pegcetacoplan in eyes 27 days after IVT injection is shown by the blue vertical line.

VH complement biomarker pairwise correlation analysis.

Pairwise correlation analysis of FI with intact complement proteins and breakdown products and ratios in the vitreous at baseline, week 36 and week 96 from subjects in cohorts 1 to 4. FI concentrations are shown by the x-axis while biomarker concentrations and ratios are shown by the y-axis. Correlations at BL are determined using log2 ng/mL concentrations, correlations at weeks 36 and 96 are determined using log2 fold change from BL. BL = Baseline; WK = week; FC = Fold Change; R = Spearman Correlation Coefficient; p = p-value.

AH complement biomarker pairwise correlation analysis.

Pairwise correlation analysis of FI with intact complement proteins and breakdown products and ratios in the aqueous humor at baseline, week 36 and week 96 from subjects in cohorts 1 to 4. FI concentrations are shown by the X-axis while biomarker concentrations and ratios are shown by the y-axis. Correlations at BL are determined using log2 ng/mL concentrations, correlations at weeks 36 and 96 are determined using log2 fold change from BL. BL = Baseline; WK = week; FC = Fold Change; R = Spearman Correlation Coefficient; p = p-value.

Modulation of C3b/iC3b and C3b/iC3b:C3 by PPY988 delivery.

(A,B) C3b/iC3b concentrations (y-axis, ng/mL) in VH samples by week post-surgery (x-axis) from subjects in cohorts 1 to 4. Mean +/- SE is shown by bars for all (black lines) broad (blue lines) and CFI RV (green lines) groups. Significant reductions in C3b/iC3b were observed at week 36 for both non-normalised (P=0.029) (A) but not total protein (TP) normalised (P=0.271) (B) data, and at week 96 for non-normalised (P=0.038) but not TP normalised (P=0.069) data. Notably these reductions are modest compared to for Ba modulation. (C,D) Ratios of C3b/iC3b:C3 concentration (Y-axis, ng/mL) were calculated for each subject at each sampling post-surgery week (X-axis). Mean +/- SE is shown by bars for all (black lines) broad (blue lines) and CFI RV (green lines) groups. No reductions were observed at week 36 for either non-normalised (P=0.173) (C) or TP normalised (P=0.588) (D) C3b/iC3b:C3 ratios. However, reductions were observed at week 96 for non-normalized (P=0.002) and TP normalized (P=0.012) C3b/iC3b:c3 ratios. Significance is defined as *P<0.05; **P<0.01, ***P<0.001. N numbers are shown above X-axis. BL = Baseline; WK= week.

Classical pathway (CP) engagement post-treatment with PPY988.

Fold-changes (y-axis) in CP complement biomarkers [C1q, C4, C4b and C4b:C4] concentrations from baseline at each post-surgery week (VH: week 5/12, 36 and 96; AH: week 36 and 96; X-axis) are shown in VH with no normalisation (A), VH with total protein normalisation (B), and AH with no normalisation. Mean is shown by thick black lines, +/- SE is shown by bars. Individual subject changes are shown by grey lines. BL = Baseline; WK= week.

Pairwise correlation analysis of vitreous versus aqueous humor complement biomarkers at baseline and post-treatment from subjects in cohorts 1 to 4.

VH concentrations on x-axis and AH concentrations on y-axis of FI, Ba, C3a and C3b/iC3b at baseline, week 36 and week 96 post-surgery. Vitreous and aqueous concentrations of FI and C3a significantly correlate at all time points, Ba concentrations correlate only at baseline, while C3b/iC3b concentrations only correlate significantly at week 36 post-treatment. BL = Baseline; WK = week; R = Spearman Correlation Coefficient; p = p-value.

Proteomic analysis of vitreous humor using Olink.

(A) Change in FI levels (Olink OID30772) over time. Median FI values measured at baseline, week 12, and week 36 are linked. Sample numbers were baseline; n=17, week 12; n=14, week 36; n=17. (B) Volcano plot of differential protein expression (week 36 relative to baseline). Minimum protein fold-change was set at absolute 1.5. Data points with a nominal p-value of less than 0.05 were considered statistically significant (red dots indicate significantly upregulated proteins, blue dots indicate significantly downregulated proteins and orange dots represent no significant change on a log2 scale).

Comparability of commercial FI to AAV2-CFI potency.

C3b alpha’ chain breakdown normalised to C3b intact beta chain (y-axis) produced by increasing concentrations of PPY988 (GMP#3) in orange compared to commercial FI (Complement Technology (CompTech), Inc) in green. The serum purified CompTech FI protein was diluted in non-transduced control sample so that both FI (reference sample) and PPY988 FI (test sample) were in the same background matrix to ensure comparability. No difference in potency was observed between PPY988 and CompTech FI Protein. Relative Potency of PPY988: 1.151; 95% CI range: 1.065-1.244; Precision: 1.168. See supplemental methods for further details.

FOCUS study overview.

FOCUS consisted of four parts of either PPY988 dose escalation or expansion, divided by seven cohorts varying according to route of gene therapy administration, dose (vg; viral genomes), and biomarker matrix. Only Parts 1 and 2 (cohorts 1 to 4) are described given subretinal gene therapy delivery permitted vitreous humour (VH) sampling. VH, aqueous humour (AH) and plasma biomarker samples were collected from subjects prior to surgery at baseline (BL), and subsequent timepoints up to week 96 post-treatment. Emerging data and analysis on cohorts 1 and 2 indicated week 5 sampling from the VH was too early to evaluate an impact post-vitrectomy, so the first post-treatment evaluation for cohorts 3 and 4 was moved to a later time-point in the study (week 12), when it was expected that the proteome would have stabilised, and the transduced cells achieved maximal expression.

Number of FOCUS samples analysed longitudinally across study.

VH and AH samples from subjects in cohorts 1 to 4 that passed acceptance criteria taken at baseline (BL) and at each timepoint post treatment are provided. Only those subjects with sampling at both BL and at least one of week 36 or 96 time-points are included.

Descriptive statistics of vitreous humour baseline complement biomarkers concentrations from subjects in cohorts 1 to 4.

The number of samples correspond to the total number which could be measured and had a value within acceptance criteria. All concentrations shown are expressed in ng/mL.

Descriptive statistics of aqueous humour baseline complement biomarkers concentrations from subjects in cohorts 1 to 4.

The number of samples correspond to the total number which could be measured and had a value within acceptance criteria. All concentrations shown are expressed in ng/mL.

Descriptive statistics of plasma baseline complement biomarkers concentrations from subjects in cohorts 1 to 4.

The number of samples correspond to the total number which could be measured and had a value within acceptance criteria. All concentrations shown are expressed in ng/mL.

Temporal change in protein levels as evaluated using the Olink platform.

Spreadsheet listing Olink (https://olink.com/) protein identifiers (IDs) that passed quality control alongside metrics for differential expression in vitreous humor comparing baseline to post-treatment at week 12 and week 36 timepoints. fdr = false discovery rate; NPX = normalised protein expression.