Preclinical safety evaluation of recombinant adeno-associated virus 2 vector encoding human tumor necrosis factor receptor-immunoglobulin Fc fusion gene

Figures & data

Table 1. The study design for preclinical safety evaluation of rAAV2/human TNFR:Fc.

Studies	Single dose	Repeat-dose	Repeat-dose
Species	Kunming mouse	Wistar rat	Rhesus Monkey
Age of animals	6 weeks	7 weeks	2–3 years
No. of animals	15/sex/group	22/sex/group	4/sex/group
Dose (Volume) of adm	7.5 × 10^12 vg/kg	1 × 10^12, 2 × 10^12, 3 × 10^12 vg/mL (50μL)	1 × 10^12, 3 × 10^12 vg/mL (1 mL)
Date of adm	Day 0	Days 0–7 (continuous 8 days) repeated adm	Days 0–7 (continuous 8 days) repeated adm
Route of adm	Intravenous	Intraarticular	Intraarticular
Recovery period	No	91 days	182 days
Parameters monitored
Clinical observations	Everyday	Everyday	Everyday
Body weight	Days 0, 1, 8, 14 and 21	Weekly	Weekly
Food consumption	ND	Weekly	Weekly
Body temperature	ND	Days 0, 1, 8, 22 and 99	Days 0, 1, 7, 20, 64, 99,134 and 190
ECG	ND	ND	Days 0, 7, 20, 43, 71, 99 and 190
Hematology	ND	Days 8, 22 and 99	Days 8, 22, 64, 99 and 190
Clinical chemistry	ND	Days 8, 22 and 99	Days 8, 22, 64, 99 and 190
Necropsy for pathology and histopathology	Day 22	Days 8, 22 and 99	Days 8, 22, 99 and 190
NTAb	ND	Days 7, 29, 71 and 99	Days 8, 36, 64, 99 and 134
Anti-hTNFR antibodies and Biodistribution analysis*	ND	Days 7, 15, 22, 29, 43, 57, 71, 85 and 99 Days 8, 22 and 99	Days 8,15, 22, 36, 64, 99 and 134 Days 8, 22, 99 and 190

Note: The saline solution (0.9% NaCl) was used as negative control in the single- and repeat-dose toxicity studies in mice, rats and monkeys. Abbreviation: adm, administration; ND, not determined. ECG, electrocardiogram; NTAb, neutralizing antibody;

* only the section tissues of rats and monkeys from high dose groups were harvested at each necropsy for the biodistribution studies.

Figure 1. Group mean body weights of rats and rhesus monkeys following the repeated administration with rAAV2/human TNFR:Fc. (A) Mean body weights of male rat; (B) Mean body weights of female rat; (C) Mean body weights of rhesus monkeys (n = 18 on study day 2, 13 on day 3–42 and 5 on day 43–84 in each gender per group for rats; n = 8 on day 2–42, and 4 on day 43–70 in each group for cynomolgus monkeys except for the animal number of saline group were halved as described in Materials and Methods, *P<0.05 vs. the saline control group).

Table 2. Antibodies to TNFR in rats and monkeys after administration with rAAV2/human TNFR:Fc.

		Number positive/number test (mean OD value ± SD)
Animal species	Day	PBS control		Low-dose group		High-dose group
Rat	7	0 /6	(0.038±0.029)	0/6	(0.028±0.018)	0/6	(0.031±0.030)
	15	0/6	(0.043±0.041)	0/6	(0.028±0.016)	0/6	(0.039±0.022)
	22	0/6	(0.057±0.043)	0/6	(0.044±0.033)	2/6	(0.371±0.556)
	29	0/6	(0.067±0.032)	0/6	(0.046±0.025)	2/6	(0.545±0.815)
	43	0/6	(0.136±0.033)	0/6	(0.083±0.028)	3/6	(0.793±0.806)
	57	0/6	(0.104±0.036)	0/6	(0.096±0.061)	5/6	(1.025±0.531)
	71	0/6	(0.135±0.044)	1/6	(0.172±0.209)	6/6	(1.181±0.506)
	85	0/6	(0.217±0.071)	1/6	(0.231±0.327)	5/6	(1.252±0.512)
	99	0/6	(0.231±0.093)	1/6	(0.290±0.413)	6/6	(1.328±0.525)
Monkey	8	0/8	(0.006±0.022)	0/8	(0.006±0.016)	0/8	(0.012±0.019)
	15	0/6	(0.004±0.007)	0/6	(0.011±0.009)	0/6	(0.003±0.020)
	22	0/6	0.000	0/6	0.000	0/6	(0.01±0.014)
	36	0/4	0.000	0/4	(0.001±0.011)	0/4	(0.047±0.014)
	64	0/4	(0.029±0.027)	0/4	(0.038±0.033)	0/4	(0.160±0.066)
	99	0/4	(0.060±0.082)	0/4	(0.071±0.059)	0/4	(0.150±0.093)
	134	0/2	(0.198±0.023)	0/2	(0.162±0.083)	0/2	(0.096±0.113)

Notes: Low- and high- dose group presents the animal were treated repeatedly with the 1 × 10¹² vg/mL and 3 × 10¹² vg/mL rAAV2/human TNFR:Fc, respectively.

Table 3. Antibodies to AAV2 in rats and monkeys after administration with rAAV2/human TNFR:Fc.

		Rat				Monkey
Group	Dilution of sera	Day7	Day29	Day71	Day99	Day8	Day36	Day64	Day99	Day134
PBS control	1:50	0/6	0/6	0/6	0/6	0/8	0/4	0/4	0/4	0/2
Low-dose		0/6	6/6	5/6	1/6	0/8	0/4	0/4	0/4	0/2
High-dose		0/6	6/6	5/6	1/6	0/8	0/4	0/4	0/4	0/2
PBS control	1:200	0/6	0/6	0/6	0/6	0/8	0/4	0/4	0/4	0/2
Low-dose		0/6	2/6	1/6	0/6	0/8	0/4	0/4	0/4	0/2
High-dose		0/6	4/6	1/6	0/6	0/8	0/4	0/4	0/4	0/2
PBS control	1:800	0/6	0/6	0/6	0/6	0/8	0/4	0/4	0/4	0/2
Low-dose		0/6	0/6	0/6	0/6	0/8	0/4	0/4	0/4	0/2
High-dose		0/6	0/6	0/6	0/6	0/8	0/4	0/4	0/4	0/2

Notes: Low- and high- dose group presents the animal were treated repeatedly with the 1×10¹² vg/mL and 3×10¹² vg/mL rAAV2/human TNFR:Fc, respectively.

Figure 2. Biodistribution of AAV2 DNA in different tissues of rats and rhesus monkeys following the repeated administration with rAAV2/human TNFR:Fc. Part tissues of rats (n = 6) and monkeys (n = 2) from high dose groups were harvested for biodistribution analysis at each necropsy. Each symbol represents the value for an individual animal. LOD represents lower limit of detection.