Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector

Abstract

For genetic diseases that manifest at a young age with irreversible consequences, early treatment is critical and essential. Neonatal gene therapy has the advantages of achieving therapeutic effects before disease manifestation, a low vector requirement and high vector-to-cell ratio, and a relatively immature immune system. Therapeutic effects or long-term rescue of neonatal lethality have been demonstrated in several animal models. However, vigorous cell proliferation in the newborn stage is a significant challenge for nonintegrating vectors, such as adeno-associated viral (AAV) vector. Slightly delaying the injection age, and readministration at a later time, are two of the alternative strategies to solve this problem. In this study, we demonstrated robust and efficient hepatic gene transfer by self-complementary AAV8 vector in neonatal mice. However, transduction quickly decreased over a few weeks because of vector dilution caused by fast proliferation. Delaying the injection age improved sustained expression, although it also increased neutralizing antibody (NAb) responses to AAV capsid. This approach can be used to treat genetic diseases with slow progression. For genetic diseases with early onset and severe consequences, early treatment is essential. A second injection of vector of a different serotype at a later time may overcome preexisting NAb and achieve sustained therapeutic effects.

FIG. 1.

Robust but unstable expression of EGFP in liver after temporal vein injection of scAAV8 vector into neonatal mice. Newborn mice (1 day old) were injected via the temporal vein with 5×10¹⁰ GC of AAV2/8sc.TBG.EGFP. Liver was harvested 1, 2, 3, 4, 5, 7, 9, 14, 21, and 35 days postinjection for evaluation of EGFP expression. Representative images from each group are shown. Scale bar: 200 μm.

FIG. 2.

Correlation of gene expression, vector genomes in liver, and body and liver growth in neonatal mice after temporal vein injection of scAAV8 vector. Newborn mice (1 day old) were injected via the temporal vein with 5×10¹⁰ or 2×10¹¹ GC of AAV2/8sc.TBG.EGFP. Livers were harvested 1, 2, 3, 4, 5, 7, 9, 14, 21, and 35 days postinjection. Transduction efficiency was evaluated by quantitative morphometric analysis of the intensity of the green fluorescence (A) and percent transduction of hepatocytes (B). Means and SD are shown (n=3–9 per time point). The horizontal dashed line indicates the background intensity value for untransduced neonatal liver. *Significant difference between the two dose groups (p<0.05, unpaired t test, two-tailed). (C) Vector genome copies per diploid genome in liver at the indicated time points as measured by Q-PCR. (D) Rate of body and liver growth in neonatal mice after vector administration.

FIG. 3.

Delaying the age at which vector was injected increases the persistence of gene expression in liver. Mice at the age of 1 day (NB) or 1–6 weeks (1W–6W) were injected intraperitoneally with 5×10¹⁰ GC of AAV2/8.TBG.EGFP. EGFP expression was evaluated 6 weeks after vector administration (A) or at the age of 12 weeks (B). Representative pictures from each group are shown. Scale bar: 200 μm. (C) Quantitative morphometric analysis of transduction efficiency, based on percent transduction of hepatocytes. (D) Vector genome copies per diploid genome in liver 6 weeks after vector administration or at the age of 12 weeks, measured by Q-PCR. Error bars represent the standard deviation (n=3–5).

FIG. 4.

Lower AAV8 NAb titer in mice injected at a younger age. Mice at the age of 1 day (NB) or 1–6 weeks (1W–6W) were injected intraperitoneally with 5×10¹⁰ GC of AAV2/8.TBG.EGFP. Serum was obtained 6 weeks after vector injection for measurement of AAV8 NAb titer.

FIG. 5.

Efficient liver transduction by AAVrh.10 vector in mice preinjected with AAV8 vector at the newborn stage. Newborn mice (1 day old) were injected via the temporal vein with 5×10¹⁰ GC of AAV2/8sc.TBG.mOTC vector, and 4 weeks later received a second injection, via the tail vein, of 1×10¹¹ GC of AAVrh.10sc.TBG.EGFP vector. Control animals received no vector injection as newborns, only AAVrh.10 at week 4. Livers were harvested 4 weeks after the second injection for evaluation of EGFP transduction. (A) EGFP expression in the liver of control mice (naive). (B) EGFP expression in the liver of mice that received AAV2/8 vector as newborns (AAV8-inj). Scale bar: 200 μm. (C) Morphometric analysis of the transduction efficiency, based on percent transduction of hepatocytes. Error bars represent the standard deviation (n=4 for naive group; n=23 for AAV8-inj group). (D) AAV NAb titer at 28 days after the first injection of AAV2/8 vector. Serum was obtained 28 days after the AAV2/8 vector injection for measurement of AAV8 and AAVrh.10 NAb titers. Because of the limited volume of the serum samples, the limit of detection of AAV8 NAb was set at 1:20 (samples with titers<1:20 are plotted as 1:10), and the limit of detection of AAVrh.10 was set at 1:5 (samples with titers<1:5 are plotted as 1:1). Each horizontal line indicates the mean value of the group (n=23).