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.