Methods
Data source: Preclinical and clinical transgene product expression data of vectors were collected from published literature. Only vectors with clinical data and preclinical data from ≥ 2 animal species available were collected. When the body weight of individual subject or animal was not available in literature, the body weights of mouse, cynomolgus macaque, rhesus macaque, and human were assumed as 0.02, 2.5, 8.0, and 70 kg, respectively.
Targeted plasma transgene product levels : The FIH dose is anticipated to be effective in eliminating uncontrolled bleeds. Thus, 12% of normal plasma FVIII activity level and 5% of normal plasma FIX level were proposed as the targeted transgene protein levels for hemophilia A and B GT, respectively.9 The normal plasma level of FIX in healthy subjects is 5000 ng/mL10 and thus a targeted FIX level of 250 ng/mL was used to project FIH doses of FIX vectors except for SPK-9001 vector encoding FIX-Pauda. FIX-Padua is a hyperfunctional variant of FIX and the residue 338 in human FIX is changed from arginine to alanine. FIX-Pauda encoded by SPK-9001 has an 8- to 12-fold increased specific activity (a mean difference of 9.1-fold in hemophilia B mice) compared to endogenous FIX.11 Therefore, the targeted plasma FIX-Pauda level was selected as 250 ng/mL ÷ 9.1 = 27.5 ng/mL (equivalent to 5% of normal FIX level).
Interspecies normalization of dose-response and FIH dose prediction : Transgene product amount in the blood circulation of individual animal and human was normalized using Equation 1:
\(Normalized\ protein={(Cprotein\ \times Blood\ volume\ \times Body\ weight)}^{0.25}\)Equation 1
Cprotein was the maximum plasma concentration of transgene product following GT. The blood volume of mouse, dog, cynomolgus macaque, rhesus macaque, and human was 79, 86, 65, 54, and 70 mL per kg body weight.12
A power regression model was used to correlate normalized transgene product amounts and the total vector doses (vg) received by different species. The regression was conducted using the data of three species (two animal species and human) and two animal species only. The targeted transgene product level (12 IU/dL for FVIII, 250 ng/mL for FIX, and 27.5 ng/mL for FIX-Pauda) was normalized using Equation 1. The normalized transgene product amount in patient’s blood circulation at the targeted level was then incorporated into the power equation derived from two preclinical species to calculate FIH dose.
GEF calculation and allometric scaling : Individual animal and human GEF values were calculated using Equation 2 previously reported by Tang et. al..5
\(GEF=Cprotein\ \times CLprotein\ \div Dose\) Equation 2
Cprotein was the maximum plasma concentration (Cmax) of transgene product following GT. CLprotein was the total clearance of FIX or FVIII, which was the average clearance values from multiple animal/human pharmacokinetic studies (supplemental Table S1). Dose was the total vector genomes (vg) received by individual animal or patient. The mean GEF of each species was used for allometric scaling. A linear regression was conducted between logGEF and logW using the data of three species (two animal species and human) and two animal species only.5 The equation derived from two animal species was used to predict human GEF.
When the human GEF predicted by allometric scaling was available, Equation 3 was used to predict FIH dose.
\(Dose=Targeted\ Cprotein\ \times CLprotein\ \div GEF\) Equation 3
Where the targeted Cprotein was 12 IU/dL for FVIII and 250 ng/mL for FIX (27.5 ng/mL for FIX-Pauda). GEF is the predicted human GEF derived from allometric scaling of two animal species.
Direct vg/kg conversion : A linear regression was conducted between monkey or dog plasma FIX levels or FVIII activity levels at various doses and total vector doses (vg). Then, plasma FIX level of 250 ng/mL (27.5 ng/mL for FIX-Pauda) or plasma FVIII activity level of 12 IU/dL was incorporated into the regression equation to estimate monkey or dog total vector dose which was anticipated to produce transgene product in monkeys or dogs at targeted level. The total dose was divided by animal body weight and the weight normalized dose (vg/kg) was the FIH dose derived from the direct vg/kg conversion approach.