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. 2013 Oct;20(10):987-96.
doi: 10.1038/gt.2013.22. Epub 2013 Jun 13.

Role of antigen-specific regulatory CD4+CD25+ T cells in tolerance induction after neonatal IP administration of AAV-hF.IX

Y Shi  1 , R Falahati, J Zhang, L Flebbe-Rehwaldt, K M L Gaensler
Affiliations
Free PMC article

Role of antigen-specific regulatory CD4+CD25+ T cells in tolerance induction after neonatal IP administration of AAV-hF.IX

Y Shi et al. Gene Ther. 2013 Oct.
Free PMC article

Abstract

Neonatal AAV8-mediated Factor IX (F.IX) gene delivery was applied as a model for exploring mechanisms of tolerance induction during immune ontogeny. Intraperitoneal delivery of AAV8/ Factor IX (hF.IX) during weeks 1-4 of life, over a 20-fold dose range, directed stable hF.IX expression, correction of coagulopathy in F.IX-null hemophilia B mice, and induction of tolerance to hF.IX; however, only primary injection at 1-2 days of life enabled increasing AAV8-mediated hF.IX expression after re-administration, due to the absence of anti-viral capsid antibodies. Adoptive splenocyte transfer from tolerized mice demonstrated induction of CD4(+)CD25(+) T regulatory (T(reg)) populations that specifically suppressed anti-hF.IX antibody responses, but not responses to third party antigen. Induction of hF.IX antibodies was only observed in tolerized mice after in vivo CD4(+)CD25(+) cell depletion and hF.IX challenge. Thus, primary injection of AAV during a critical period in the first week of life does not elicit antiviral responses, enabling re-administration of AAV and augmentation of hF.IX levels. Expansion of hF.IX-specific CD4(+)CD25(+) T(regs) has a major role in tolerance induction early in immune ontogeny. Neonatal gene transfer provides a useful approach for defining the ontogeny of immune responses and may suggest approaches for inducing tolerance in the context of genetic therapies.

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Figures

Figure 1
Figure 1
Long term, therapeutic hF.IX levels without induction of humoral responses after IP delivery of AAV8-hF.IX. BALB/c mice received IP injection of 1.6 × 1010 vg per g AAV8-hF.IX at 1–2 day of life (n=25). Blood samples were collected beginning 4 weeks after AAV8-hF.IX delivery. (a) Human F.IX levels in plasma were quantified by ELISA. (b) ELISA of anti-hF.IX antibody titers. Positive control adults were produced by IP injection of 10 μg purified hF.IX/Alum. Negative controls included plasma from AAV8-Luc IP injected mice. Naive control plasma samples were from uninjected adult mice. (c) ELISA for anti-AAV8 capsid antibody. Positive control samples were produced by IP injection of adult mice with 5 × 109 Vg per g AAV8-Luc virus/Alum. The negative control for anti-AAV assay was the plasma from hF.IX/Alum-injected mice. ELISA of plasma samples from naive animals is also shown.
Figure 2
Figure 2
Dose-dependent hF.IX expression after neonatal IP administration of AAV8-hF.IX without induction of anti-hF.IX antibodies. After IP injection of BALB/c neonates with increasing dosages of AAV8-hF.IX (1.6–32 × 109 vg per g) and adults at 1.6 × 109 vg per g, serial blood samples were collected beginning 4 weeks after AAV8-hF.IX delivery. (a) Human F.IX levels in plasma were quantified by ELISA as described. (b) ELISA of anti-hF.IX antibody titers after different IP doses of AAV8-hF.IX. The viral dose × 109 vg per g is indicated for each group under individual columns. Samples from injected neonates or adults are indicated. Positive control (+) was plasma from hF.IX/Alum injected adults. Negative control (−) was plasma from AAV8-Luc injected mice. ELISA of plasma from naive animals is also shown. (c) ELISA for anti-AAV8 capsid antibody. Experimental groups are the same as described in Figure 2b. Positive control (+) was plasma from adult mice injected with 5 × 109 vg per g AAV8-Luc/Alum. Negative control (−) was plasma from hF.IX/alum-injected animals. ELISA of plasma from naive mice is shown at far right.
Figure 3
Figure 3
Augmented hF.IX levels with AAV8-hF.IX re-administration only observed in animals receiving primary injection at day 2 of life. BALB/c mice received primary injection with 1.6 × 109 vg per g of AAV8-hF.IX at the indicated ages (1–2 day of life, 1, 2 3 weeks or adult). Secondary IP injection of AAV8-hF.IX was performed 9 weeks after the primary injection in all groups. All groups were then challenged with injection of hF.IX/Alum 17 weeks after secondary AAV8-hF.IX administration. (a–e) hF.IX levels were initially quantified in plasma by ELISA, 4 weeks after primary injection, and serially thereafter up to 40 weeks. (f–h) anti-hF.IX antibody levels. (f) 8 weeks after primary injection; (g) 4 weeks after secondary AAV8-hF.IX IP injection; (h) 4 weeks after challenge with hF.IX/Alum. The age of mice at primary AAV8-hF.IX injection is indicated below each column. Positive (+), Negative (−) and naive controls are as described in Figure 2b.
Figure 4
Figure 4
Critical period for IP AAV administration without anti- AAV8 capsid after IP injection of AAV-hF.IX. Primary IP injection of 1.6 × 109 vg per g of AAV8-hF.IX in BALB/c mice at different ages (1–2 day of life, 1, 2 3 weeks of life or as adults) was followed by re-administration of AAV8-hF.IX 9 weeks later. Serial blood samples were collected beginning 4 weeks after primary AAV8-hF.IX delivery for ELISA to detect anti-AAV8 capsid antibodies. Positive, negative and naive controls were as described in Figures 1c. (a) Anti-AAV8 capsid titers 4 weeks after primary injection (b) Anti-AAV8 capsid titers 4 weeks after secondary injection of AAV8-hF.IX (13 weeks after primary injection).
Figure 5
Figure 5
Effect of adoptive transfer of splenocyte populations from AAV8-hF.IX neonatal injected mice on immune responses to hF.IX/Alum and Luc/Alum challenge. Spenocytes from naive adult mice or adult mice after neonatal AAV8-hF.IX delivery, were infused IV, 24 h before challenge with hF.IX/Alum or Luc/Alum. Total splenocytes (5 × 107), purified CD4+ T cells (1 × 107), CD4- cell (5 × 107), CD4+CD25+ cells (1 × 106) or CD4+CD25- T cells (9 × 106) were transferred. Each bar represents the average antibody titer for 5 animals (±s.d.). (a) Anti-hF.IX antibody titer. (b) The level of anti-Luc antibody 2 weeks after immunologic challenge. Response to third party antigen was tested by injection of luciferase/Alum 24 h after adoptive transfer of splenocyte populations, as described in (a).
Figure 6
Figure 6
Phenotypic correction of F.IX knockout mice in CD-1 strain without induction of anti-hF.IX or anti-AAV8 capsid antibodies after delivery of AAV8-hF.IX on 1–2 day of life. Treatment of hemophilia B (CD-1 F9−/− designated as CD-1 FIX KO) mice by neonatal IP AAV8-hF.IX gene transfer (3.2 × 109 Vg per g) (n=10). Shown are: (a) systemic hF.IX levels. (b) the level of anti-hF.IX antibodies 4 weeks after vector administration, (c) measurement of anti-AAV8 capsid antibody titers, and (d) measurement of coagulation time by aPTT, 4 weeks after IP vector administration.
Figure 7
Figure 7
Tissue bio-distribution analysis of mice injected neonatally IP with AAV8-hF.IX in neonates. Mice were injected on day 1–2 of life doses of 5 × 109 vg per g for AAV8-hF.IX. Tissues were harvested from neonatally injected mice at 20 weeks after vector administration. Real-time PCR was performed for detecting the number of copies in 200 ng of genomic DNA, and the data is displayed as the number of hF.IX copies per μg gDNA. Negative controls are tissue from naive BALB/c mice.
Figure 8
Figure 8
RT-PCR analysis of hF.IX mRNA expression in mice neonatally administrated with AAV8-hFIX IP. Mice were sacrificed 10 weeks after a single neonatal injection. Tissues were harvested and total RNA was extracted and amplified by RT-PCR. First lane on left: marker; Five lanes on left show RT-PCR products from an un-injected, age-matched, naive control mouse. The five lanes on the right represent RT-PCR products from tissues harvested after IP neonatal injection of AAV8-hF.IX. Tissues analyzed include heart, diaphragm, liver, muscle from lower extremity, and peritoneum with underlying muscle.
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