1. DilutionDilution
AAV 2/8.eGFP
Lamb #2003
Abigail Andrews and Marcus Davey, PhD
The Children’s Hospital of Philadelphia, The Center for Fetal Research
IntroductionIntroduction
The Next Step in Curing Genetic Disease
Data / ResultsData / Results
ReferencesReferences
AcknowledgementAcknowledgement
Gene therapy is the transplantation of corrected genes into cells to replace defective
or missing genes for the treatment of genetic disorders. Viral vectors, engineered to
act as a carrying mechanism, can deliver and insert the corrected genes (termed
transgenes) into a targeted cell thereby correcting the genetic disorder.
Although a promising treatment option for a number of heritable diseases, host
immunological responses to the therapeutic virus and transgene remain a major
obstacle for postnatal gene therapy, particularly for diseases that require repeated
vector administration1
.
Experimental data suggests that such immunological barriers may be overcome by
inducing “immunological tolerance” to viral capsid proteins and transgene product in
the period during fetal development in which the thymus is undergoing education of
self proteins – identifying what is “self” and “non-self”.2
The introduction of foreign
proteins during this period of fetal thymic education (14-18 weeks gestation in
humans) ensures that the foreign proteins will be recognized as non-foreign and thus
that no humoral response will be mounted. Theoretically, induction of
immunological tolerance to therapeutic viruses and their transgenes during fetal life
would enable repeated postnatal administration of viral vectors without the patient
mounting an immune response.
Thank you to the members of the Children’s Hospital of Philadelphia, Center for Fetal
Research, specifically Dr. Marcus Davey and Dr. Alan Flake. Thank you to the faculty
of the Baldwin Science Department, specifically Mrs. Christie Reed, Mr. Nick Vechik,
and Dr. Susan Dorfman.
As the promise of Adeno-associated viral -based gene therapy becomes increasingly
realized, safe strategies to overcome both preexisting and induced host immune
responses to vector capsid antigens and transgene product are required to improve
therapeutic efficacy. This challenge is significant for genetic diseases which may require
repeated administration of viral vector to boost transgene expression. In this large animal
study, we demonstrate that postnatal tolerance to a foreign protein (ie. GFP) can be
achieved via intravascular AAV-IUGT to mid-gestation fetal sheep.
Figure 1 displays the hepatic expression of GFP (fluorescence and immunohistochemisty;
IHC) in fetal lambs at 1 month post-injection that received intravascular AAV2/8 and
AAV2/6. The presence of GFP in these biopsies confirmed the durability of of the
transgene (GFP) expression.
Figure 2 displays the ELISA analysis of serum anti-GFP antibodies before (Pre-challenge)
and three weeks (Week 3) after intramuscular immunization with AAV2/1.eGFP in
neonatal lambs (~ 1 month of age) that received in utero (~65 days gestation)
intravascular injection of AAV2/8 (A) or AAV2/6.2 (E) expressing GFP transgene. At 6
months of age, GFP expression was assessed in hind-limb muscle of the same lambs;
AAV2/8 (B,C,D), AAV2/6.2 (F,G,H). A control animal that did not receive in utero gene
therapy (IUGT) generated a robust humoral (IgG) immune response to GFP (Panel I) at 3
weeks following intramuscular injection of AAV2/1.eGFP, and demonstrated significant
neutrophil influx at the muscle injection site (J,K,L).
In view of our results, it is conceivable that AAV-IUGT could be harnessed to induce
tolerance to therapeutic vectors via in utero administration of AAVs engineered to express
viral capsid antigens known to illicit postnatal immune responses. Theoretically, this
would permit repeated postnatal administration of viral vector while avoiding host
immune responses to vector antigens and the need for immunosuppression prior to
vector administration. Although a promising and exciting concept, additional studies in
the large animal model are needed to assess efficacy and safety of inducing tolerance to
viral vectors via AAV-IUGT.
MethodsMethods
Research was performed using a preclinical, fetal sheep model. Adeno-associated
viral (AAV) vectors AAV2/8 and AAV2/6 expressing green fluorescent protein3
(GFP; a
foreign protein) were administered intravenously to fetal sheep (#2003, “Wiz” and
#465, “Cupid”) during mid-gestation at ~65 days (full term is ~150 days in sheep). At
approximately 1 month after birth (~110 days post-injection), a liver wedge biopsy
was performed under general inhalation anesthesia. Hepatic GFP expression was
assessed by fluorescence microscopy and GFP immunohistochemistry of formalin-
fixed, paraffin-embedded sections (3-5µm)4
. At ~2 months after birth, lambs
underwent an immunological challenge with GFP to assess whether immunological
tolerance had been achieved; this was performed via an intramuscular injection of
AAV2/1.eGFP. Serum samples were then collected at weekly intervals up to 5 weeks
post-injection. ELISA assays were performed to measure antibody responses to GFP.
Animals were euthanized at ~6 months of age (adult age) and postmortem organ
analysis was performed to confirm the expression or lack thereof of GFP.
Discussion / ConclusionsDiscussion / Conclusions
Pre-immune Fetus
Tolerization to
capsid
proteins/transgen
es
Repeated post-natal vector
administration
Transgene Expression
Correction
of genetic
disorder
~ 65 days IV ~150 days
AAV2/8.eGFP
&
AAV2/6.2.eGFP
Birth
~4 weeks
Liver
Biopsy
~2 months
Immune
Challenge
AAV2/1.eGFP
Serum samples collected
~6 Months
Euthanasia
Figure 1 - Liver Biopsy Results:
AAV 2/8.eGFP
Lamb #2003
(GFP-immunoperoxidase, 4um)
AAV 2/6.eGFP
Lamb #465
(3.22x10^12 gc)
(1.66x10^11 gc)
Figure 2 – ELISA Analysis of Serum Samples and Postmortem Organ Analysis
AAV 2/6.eGFP
Lamb #465
1
Amado, D., Mingozzi, F., Hui, D., Bennicelli, J. L., Wei, Z., Chen, Y., . . . Bennett, J. (2010) Sci Transl
Med, 2(21), 21ra16. doi: 10.1126/scitranslmed.3000659
2
Flake, A. W., Harrison, M. R., Adzick, N. S., & Zanjani, E. D. (1986) Science, 233(4765), 776-778
3
Joyeux, L., Danzer, E., Limberis, M. P., Zoltick, P. W., Radu, A., Flake, A. W., & Davey, M. G. (2014)
Hum Gene Ther Methods, 25(3), 197-205. doi: 10.1089/hgtb.2013.143
4
Davey, M. G., Zoltick, P. W., Todorow, C. A., Limberis, M. P., Ruchelli, E. D., Hedrick, H. L., & Flake, A.
W. (2012) Gene Ther, 19(2), 201-209. doi: 10.1038/gt.2011.83
Adeno-Associated Virus
Molecular Model
Control
(No IUGT)