In these studies, intraperitoneal delivery of AAV8-hF.IX in neonates in the first month of life produced dose-dependent, stable levels of hF.IX expression. No humoral responses to hF.IX were detected after primary delivery of AAV8-F.IX in the dose range administered, or after challenge with hF.IX protein and adjuvant. Adoptive transfer and in vivo
depletion studies demonstrated that tolerance is mediated early in immune ontogeny, at least in part, by the induction of antigen-specific CD4+
. Other F.IX gene transfer studies have focused on IM or IH delivery of AAV in adults.42, 45
However, IM injections have been more likely to induce humoral responses than have IV or IH injections, even early in immune ontogeny.24
In studies by others, neonatal IP administration of AAV5-CMV-hF.IX produced stable, albeit lower levels of hF.IX than were achieved here with AAV8-hF.IX.32
In contrast to our IP delivery studies showing stable expression of the secreted hF.IX protein for >1 year, decreasing F.IX expression was observed in other neonatal models.46
Anti-hF.IX antibody responses described by others after IM AAV2-hF.IX injection, in utero
or in neonates,24
were not elicited in the current study after IP injection of AAV8-hF.IX. As the continuous presence of self-antigens has been shown to be required for maintenance of tolerance in prior studies with neonates,47, 48
it is likely that the stable hF.IX expression produced after neonatal IP administration is critical for the induction and maintenance of tolerance in this model.
We demonstrate a direct dose/response relationship between increasing doses of AAV8-hF.IX and hF.IX expression levels; however, induction of tolerance to hF.IX was not correlated with either the dose of AAV8-hF.IX in the dose range examined, or with levels of F.IX achieved after neonatal IP administration. This is in contrast to recent findings with delivery of AAV1-hF.IX in adult mice where induction of tolerance to hF.IX was AAV vector dose-dependent.42
In studies by Kelly et al.42
the minimum AAV1-hF.IX dose reproducibly resulting in tolerance induction in the C57BL/6 strain was 2 × 1010
vg per g. Recent in utero
IP gene delivery studies in fetal sheep using self-complementary AAV-hF.IX vectors (10 × 109
vg per g) also did not induce anti-hF.IX antibodies after primary injection; however, low levels of hF.IX were detected up to 6 months after injection with failure to induce tolerance to hF.IX.49
In our studies, tolerance to hF.IX was induced in all groups of animals, where levels of hF.IX expression were in general 20-fold higher at 6 months, than that achieved in the sheep model. Thus, not only the route of delivery and stage of immune ontogeny at which AAV is administered are critical in determining the level and nature of immune responses, but also whether sustained transgene expression levels are adequate for the maintenance of tolerance.
In this context, the immune mechanisms enabling long-term gene expression and absence of anti-capsid responses after neonatal delivery, and the time frame when this may be achieved have not been previously examined. In order to define such a critical period, primary injection at different ages (days 1–2 of life, 1, 2 or 3 weeks and adult) was tested and showed that only the group receiving days 1–2 injections exhibited a major increase in hF.IX after secondary injection. Consistent with this result, anti-AAV8 capsid antibody was not detectable in mice undergoing primary injection during the first week of life, but anti-AAV8 antibodies were detected in all other groups of mice after re-administration of AAV8-hF.IX.
A number of recent studies have addressed the challenges of establishing safe and effective routes of gene delivery, and reducing immune responses to AAV vectors in adults. Transvenular administration of F.IX in a canine model produced therapeutic levels of F.IX, albeit with anti-AAV capsid responses.50
Addition of transient immunosuppression enhanced F.IX levels and abrogated anti-F.IX antibody responses.51
In other studies, administration of proteasome inhibitors enhanced AAV-mediated transduction and decreased capsid antigen presentation.52, 53
Transient immunosuppression with Mycophenolate Mofetil (MMF) and sirolimus54
or B-cell depletion with anti-CD20 have also reduced immune responses to AAV-mediated gene expression.55
Finally the use of micro-RNA-regulated gene therapy (miR-142 targeted) has been combined with the use of tissue-specific promoters to enhance cell specificity of expression and generate tolerogenic responses.56
The development of safe immunomodulatory approaches will enable more effective AAV-mediated genetic correction of hemophilia B.
Adoptive transfer of splenocyte populations demonstrated that CD4+
cells from BALB/c mice, neonatally injected with AAV8-hF.IX, suppressed anti-hF.IX responses after hF.IX/Alum challenge. Administration of anti-CD25 antibody was used to deplete activated T cells, including Treg
transiently expressing CD25, that suppress effector T-cell responses necessary for the formation of anti-hF.IX antibody.36
Anti-CD25 antibody-mediated in vivo
depletion of CD25+
cells in mice tolerized by neonatal IP injection enabled induction of low-level anti-hF.IX antibody responses after hF.IX/Alum challenge.
The role of Treg
in specifically inhibiting immune responses and in the maintenance of self-tolerance has been investigated in several other contexts.57, 58
In adult models of AAV-mediated F.IX gene transfer, conflicting data regarding the role of Treg
has been reported after hepatic versus IM administration.36, 42, 46
Suppression of antibody formation to hF.IX was shown to be mediated by CD4+
after IH AAV-mediated hF.IX gene transfer.36
In contrast, with IM delivery of AAV1-hF.IX gene transfer, no increase in Treg
was detected in F.IX-tolerant mice and adoptive transfer of splenocytes from hF.IX-tolerant mice did not suppress anti-hF.IX immunity.42
Neither in vitro
, nor in vivo
depletion of Tregs
reversed F.IX tolerance in this setting.42, 46
Our data are consistent with the findings of Cao et al.36
with IH AAV-F.IX gene transfer in adult models, and support a major role for CD4+
in tolerance induction to hF.IX after neonatal IP gene delivery.
The development of alternative strategies to achieve long-term transgene expression while avoiding toxic and abrogative immune responses represents an important and timely objective for genetic therapy. The relevance of these studies is underscored by pre-clinical studies in non-human primates in which codon-optimized, self-complementary AAV5 and AAV8-F.IX were delivered IV-producing efficient hepatic transduction, but also stimulating serotype-specific humoral responses.59
Further defining immune mechanisms underlying tolerance induction during immune ontogeny may inform the design of novel approaches for limiting or eliminating immune responses that abrogate therapeutic gene expression.