An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells
2017; Cell Press; Volume: 5; Linguagem: Inglês
10.1016/j.omtm.2017.04.004
ISSN2329-0501
AutoresBrett Palaschak, Damien Marsic, Roland W. Herzog, Sergei Zolotukhin, David M. Markusic,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoMultiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer. Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer. Adeno-associated virus (AAV) is a nonpathogenic weakly immunogenic dependoparvovirus with a single-stranded DNA genome of ∼4.7 kb. Humans are naturally infected with AAV, often in the presence of an immunogenic helper virus, which is required for AAV replication. This dependency is thought to provide the necessary activation signals that prime anti-AAV humoral and cell-mediated immunity.1Zaiss A.K. Liu Q. Bowen G.P. Wong N.C. Bartlett J.S. Muruve D.A. Differential activation of innate immune responses by adenovirus and adeno-associated virus vectors.J. Virol. 2002; 76: 4580-4590Crossref PubMed Scopus (326) Google Scholar AAV-based vectors have been developed to episomally deliver therapeutic genes into post-mitotic cells, such as hepatocytes, and provide stable long-term expression. Patients with hemophilia B lack functional coagulation factor IX (FIX) protein and are treated by infusing recombinant or plasma-derived FIX protein. Because FIX is normally produced by hepatocytes and FIX activity levels as low as 5% of normal can significantly reduce bleeding events, gene therapy was considered a promising means of restoring FIX expression. Positive outcomes in preclinical studies evaluating liver-directed AAV vectors expressing the coagulation FIX protein, in small and large animal models,2Mingozzi F. Liu Y.L. Dobrzynski E. Kaufhold A. Liu J.H. Wang Y. Arruda V.R. High K.A. Herzog R.W. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer.J. Clin. Invest. 2003; 111: 1347-1356Crossref PubMed Scopus (376) Google Scholar, 3Mount J.D. Herzog R.W. Tillson D.M. Goodman S.A. Robinson N. McCleland M.L. Bellinger D. Nichols T.C. Arruda V.R. Lothrop Jr., C.D. High K.A. Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.Blood. 2002; 99: 2670-2676Crossref PubMed Scopus (282) Google Scholar, 4Rogers G.L. Herzog R.W. Gene therapy for hemophilia.Front. Biosci. (Landmark Ed.). 2015; 20: 556-603Crossref PubMed Scopus (40) Google Scholar prompted the first clinical trial of an AAV2 vector (expressing FIX from a hepatocyte-specific enhancer/promoter combination, AAV2-ApoE-hAAT-F9) delivered to human hepatocytes.5Manno C.S. Pierce G.F. Arruda V.R. Glader B. Ragni M. Rasko J.J. Ozelo M.C. Hoots K. Blatt P. Konkle B. et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat. Med. 2006; 12: 342-347Crossref PubMed Scopus (1575) Google Scholar A patient receiving a vector dose of 2 × 1012 vector genome (vg)/kg had transient therapeutic FIX expression levels that returned to baseline accompanied with a self-resolving transaminitis.5Manno C.S. Pierce G.F. Arruda V.R. Glader B. Ragni M. Rasko J.J. Ozelo M.C. Hoots K. Blatt P. Konkle B. et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat. Med. 2006; 12: 342-347Crossref PubMed Scopus (1575) Google Scholar It was hypothesized and later demonstrated that the activation of AAV2 capsid memory CD8+ T cells was the likely cause for the elimination of AAV2-transduced hepatocytes.5Manno C.S. Pierce G.F. Arruda V.R. Glader B. Ragni M. Rasko J.J. Ozelo M.C. Hoots K. Blatt P. Konkle B. et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat. Med. 2006; 12: 342-347Crossref PubMed Scopus (1575) Google Scholar, 6Mingozzi F. Maus M.V. Hui D.J. Sabatino D.E. Murphy S.L. Rasko J.E. Ragni M.V. Manno C.S. Sommer J. Jiang H. et al.CD8(+) T-cell responses to adeno-associated virus capsid in humans.Nat. Med. 2007; 13: 419-422Crossref PubMed Scopus (532) Google Scholar, 7Pien G.C. Basner-Tschakarjan E. Hui D.J. Mentlik A.N. Finn J.D. Hasbrouck N.C. Zhou S. Murphy S.L. Maus M.V. Mingozzi F. et al.Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors.J. Clin. Invest. 2009; 119: 1688-1695Crossref PubMed Scopus (147) Google Scholar, 8Finn J.D. Hui D. Downey H.D. Dunn D. Pien G.C. Mingozzi F. Zhou S. High K.A. Proteasome inhibitors decrease AAV2 capsid derived peptide epitope presentation on MHC class I following transduction.Mol. Ther. 2010; 18: 135-142Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar To avoid potential immunological responses to the AAV capsid, follow-up clinical studies have now adopted transient immune suppression with prednisolone, alternative capsid serotypes, and optimized F9 expression cassettes including codon optimization and a hyperactive FIX (R338L) variant.9Nathwani A.C. Rosales C. McIntosh J. Rastegarlari G. Nathwani D. Raj D. Nawathe S. Waddington S.N. Bronson R. Jackson S. et al.Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.Mol. Ther. 2011; 19: 876-885Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 10Monahan P. Walsh C.E. Powell J.S. Konkle B.A. Josephson N.C. Escobar M. McPhee S.J. Litchev B. Cecerle M. Ewenstein B.M. et al.Update on a phase 1/2 open-label trial of BAX335, an adeno-associated virus 8 (AAV8) vector-based gene therapy program for hemophilia B.J. Thromb. Haemost. 2015; 13: 87Crossref Scopus (25) Google Scholar, 11UniQure Biopharma BV, and Chiesi Farmaceutici, SpA. Trial of AAV5-hFIX in severe or moderately severe hemophilia B. National Library of Medicine. 2015, NCT02396342. https://clinicaltrials.gov/show/NCT02396342.Google Scholar, 12Spark Therapeutics, Children's Hospital of Philadelphia, University of Pittsburgh, Royal Prince Alfred Hospital, and St. James's Hospital. Hemophilia B gene therapy - Spark. National Library of Medicine. 2012, NCT01620801. https://clinicaltrials.gov/show/NCT01620801.Google Scholar, 13Spark Therapeutics, Children's Hospital of Philadelphia, The Hemophilia Center of Western Pennsylvania, and Royal Prince Alfred Hospital. LTFU for gene transfer subjects with hemophilia B. National Library of Medicine. 2007, NCT00515710. https://clinicaltrials.gov/show/NCT00515710.Google Scholar, 14Spark Therapeutics and Pfizer. A gene therapy study for hemophilia B. National Library of Medicine. 2015, NCT02484092. https://clinicaltrials.gov/show/NCT02484092.Google Scholar, 15St. Jude Children's Research Hospital, National Heart, Lung, and Blood Institute, Hemophilia of Georgia, Inc., and Children's Hospital of Philadelphia. Dose-escalation study of a self complementary adeno-associated viral vector for gene transfer in hemophilia B. National Library of Medicine. 2009, NCT00979238. https://clinicaltrials.gov/show/NCT00979238.Google Scholar, 16Baxalta US Inc. Open-label single ascending dose of adeno-associated virus serotype 8 factor IX gene therapy in adults with hemophilia B. National Library of Medicine. 2012, NCT01687608. https://clinicaltrials.gov/show/NCT01687608.Google Scholar What was particularly frustrating for the AAV gene therapy field was that no preclinical studies predicted a limiting role of capsid-specific CD8+ T cells (Cap-CD8) in long-term FIX expression. Although an absence of Cap-CD8 responses in mice was anticipated because of no prior exposure to wild-type AAV,17Herzog R.W. Immune responses to AAV capsid: are mice not humans after all?.Mol. Ther. 2007; 15: 649-650Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar nonhuman primates are naturally infected with AAV and also failed to predict this outcome.18Li H. Lasaro M.O. Jia B. Lin S.W. Haut L.H. High K.A. Ertl H.C. Capsid-specific T-cell responses to natural infections with adeno-associated viruses in humans differ from those of nonhuman primates.Mol. Ther. 2011; 19: 2021-2030Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Until recently, efforts directed toward mimicking the immunological response against AAV capsid in mouse models had been unsuccessful.19Somanathan S. Breous E. Bell P. Wilson J.M. AAV vectors avoid inflammatory signals necessary to render transduced hepatocyte targets for destructive T cells.Mol. Ther. 2010; 18: 977-982Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 20Siders W.M. Shields J. Kaplan J. Lukason M. Woodworth L. Wadsworth S. Scaria A. Cytotoxic T lymphocyte responses to transgene product, not adeno-associated viral capsid protein, limit transgene expression in mice.Hum. Gene Ther. 2009; 20: 11-20Crossref PubMed Scopus (14) Google Scholar, 21Li H. Lin S.W. Giles-Davis W. Li Y. Zhou D. Xiang Z.Q. High K.A. Ertl H.C. A preclinical animal model to assess the effect of pre-existing immunity on AAV-mediated gene transfer.Mol. Ther. 2009; 17: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 22Wang L. Figueredo J. Calcedo R. Lin J. Wilson J.M. Cross-presentation of adeno-associated virus serotype 2 capsids activates cytotoxic T cells but does not render hepatocytes effective cytolytic targets.Hum. Gene Ther. 2007; 18: 185-194Crossref PubMed Scopus (93) Google Scholar, 23Li H. Murphy S.L. Giles-Davis W. Edmonson S. Xiang Z. Li Y. Lasaro M.O. High K.A. Ertl H.C. Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes.Mol. Ther. 2007; 15: 792-800Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 24Li C. Hirsch M. Asokan A. Zeithaml B. Ma H. Kafri T. Samulski R.J. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.J. Virol. 2007; 81: 7540-7547Crossref PubMed Scopus (80) Google Scholar Studies conducted in mice with an AAV capsid engineered to express the CD8+ epitope for ovalbumin (ova) induced activated ova-specific CD8+ T cells, confirming that input AAV capsid was processed and presented in mice.25Li C. Hirsch M. DiPrimio N. Asokan A. Goudy K. Tisch R. Samulski R.J. Cytotoxic-T-lymphocyte-mediated elimination of target cells transduced with engineered adeno-associated virus type 2 vector in vivo.J. Virol. 2009; 83: 6817-6824Crossref PubMed Scopus (38) Google Scholar Therefore, we hypothesized that previous attempts may have lacked specific and sufficient activation signals during capsid antigen exposure. To address this, we designed an immunization protocol with an in vivo prime and boost followed by in vitro expansion. In place of full-length capsid, we used the dominant capsid CD8+ T cell epitope,26Sabatino D.E. Mingozzi F. Hui D.J. Chen H. Colosi P. Ertl H.C. High K.A. Identification of mouse AAV capsid-specific CD8+ T cell epitopes.Mol. Ther. 2005; 12: 1023-1033Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar a CpG oligonucleotide activator of TLR9, and CD8+ T cell immune-stimulatory cytokines (interleukin-2 [IL-2], IL-15, and IL-21) to expand and activate Cap-CD8 followed by adoptive transfer into immune-deficient BALB/c-Rag1−/− mice.27Martino A.T. Basner-Tschakarjan E. Markusic D.M. Finn J.D. Hinderer C. Zhou S. Ostrov D.A. Srivastava A. Ertl H.C. Terhorst C. et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (119) Google Scholar Although this model reflected the elimination of AAV-transduced hepatocytes, as observed in human clinical trials, immune-deficient mice come with a number of limitations. The following studies were designed to improve the utility of the model by transitioning into immune-competent mice. We observed a comparable elimination of AAV2-ApoE-hAAT-F9-transduced hepatocytes by Cap-CD8 and then proceeded to examine the immunogenicity of two engineered library-selected mouse hepatotropic AAV2 variants. Our initial Cap-CD8 model was developed using immune-deficient Rag1−/− mice that lacked endogenous B and T cells,27Martino A.T. Basner-Tschakarjan E. Markusic D.M. Finn J.D. Hinderer C. Zhou S. Ostrov D.A. Srivastava A. Ertl H.C. Terhorst C. et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (119) Google Scholar based on the speculation that a lymphopenic environment was required to support engraftment of adoptively transferred Cap-CD8. Yet, the use of an immune-deficient mouse model has inherent limitations. Therefore, we investigated whether the Cap-CD8 model would function in immune-competent mice. Using the CD90 (Thy-1) surface antigen, expressed as either CD90.1 or CD90.2 (Thy-1.1 or Thy-1.2) on T cells, allowed us to track adoptively transferred CD90.2-Cap-CD8 into congenic CD90.1-BALB/c mice (Figure 1A). For this study, we selected the AAV2-ApoE-hAAT-F9 vector at a dose of 1 × 1011 vg, where Cap-CD8 eliminated transduced hepatocytes and reduced circulating human FIX (hFIX) levels in Rag1−/− mice.27Martino A.T. Basner-Tschakarjan E. Markusic D.M. Finn J.D. Hinderer C. Zhou S. Ostrov D.A. Srivastava A. Ertl H.C. Terhorst C. et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (119) Google Scholar Mice receiving Cap-CD8 compared with controls had a significant reduction in circulating hFIX levels at all time points (Figure 1B). Alanine aminotransferase (ALT) levels in plasma, a sensitive marker for liver damage, were measured at each time point (Figure 1C). Unlike the Rag1−/− mouse Cap-CD8 model in which ALT was elevated at 1 week post Cap-CD8 transfer, we observed an elevation in ALT (∼10-fold over normal levels of 9.3 ± 4.3 U/L) at 2 (in two of five mice) and 3 weeks (in one of five mice) following Cap-CD8 adoptive transfer. In all cases, ALT levels returned to baseline the following week. It is interesting to note that in the immune-competent Cap-CD8 model, the time course for transaminitis from Cap-CD8 targeting AAV2-transduced hepatocytes correlates better to that observed in a human patient. To confirm the specificity of Cap-CD8 elimination of AAV2-transduced hepatocytes in immune-competent mice, we performed a control study using an irrelevant AH1 gp70 epitope SPSYVYHQF to generate control CD8+ T cells (Con-CD8). Con-CD8 were induced, expanded, and adoptively transferred into BALB/c recipient mice following the same protocol as depicted in Figure 1A. Notably, there was no reduction in hFIX levels in Con-CD8 (Figure 1D, purple tracing) compared with AAV2 controls (Figure 1D, blue tracing) at all time points. Weekly monitoring of ALT levels failed to detect any elevation above what we have previously measured in AAV2-only-treated controls (Figures 1C and 1E). Overall, these data demonstrate that the reduction in hFIX levels was specific to Cap-CD8 transfer. Liver tissue was collected from mice in each group at 1 and 4 weeks after gene transfer, and cryosections were stained using antibodies against hFIX (red) and CD90.2+ T cells (green). Representative liver sections at 40× magnification from Cap-CD8 and control mice at 1 and 4 weeks post gene transfer are shown in Figure 2A. The percent of hFIX+-expressing hepatocytes was averaged from multiple images at 200× magnification using Volocity software. Mice receiving Cap-CD8 had a significant reduction in hFIX+ hepatocytes at both 1 and 4 weeks post gene transfer (Figure 2B), reflecting the reduction in circulating hFIX in plasma (Figure 1B). We were unable to detect infiltrating CD90.2+ cells in livers collected from Cap-CD8 mice at either 1 or 4 weeks, in contrast with our previous studies in immune-deficient mice,27Martino A.T. Basner-Tschakarjan E. Markusic D.M. Finn J.D. Hinderer C. Zhou S. Ostrov D.A. Srivastava A. Ertl H.C. Terhorst C. et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (119) Google Scholar although it should be noted that Cap-CD8 were sparse in liver tissue. One likely explanation is that the absence of lymphocytes in the livers of Rag1−/− mice provides a niche for long-term residence of Cap-CD8 cells, whereas this is likely filled by endogenous CD8+ T cells in immune-competent mice. Nonetheless, flow cytometry analysis performed on splenocytes isolated from controls (CD90.2+-BALB/c mice) and Cap-CD8 adoptive transfer mice (CD90.1+-BALB/c mice) at week 4 revealed a small, but distinct fraction of CD3+ CD8+ CD90.2+ T cells in Cap-CD8-treated mice (Figure 3). Thus, the loss in both circulating and hepatocyte hFIX expression, elevation in ALT, and flow cytometry analysis demonstrates that a lymphopenic host is not required for the short-term persistence and functionality of adoptively transferred Cap-CD8.Figure 3Cap-CD8 Persist in Immune-Competent Mice up to 4 Weeks after Adoptive TransferShow full captionCongenic BALB/c mice, CBy.PL(B6)-Thy1a/ScrJ, were used, in which their T cells express the CD90.1 (Thy1.1) surface antigen to track adoptively transferred Cap-CD8 generated in CD90.2 (Thy1.2) donor mice. (A) Gating strategy of antibody-labeled splenocytes stained with anti-mouse CD3-BV421, B220-BV605, CD8-A488, and CD90.2-APC. CD90.2+ CD8+ T cell gating was determined using a fluorescence minus one (-CD90.2-APC)-stained sample. (B) Representative dot plots of stained splenocytes from four BALB/c-CD90.1 (CD90.2-Cap-CD8) and BALB/c-CD90.2 control mice at 4 weeks following Cap-CD8 adoptive transfer. Left box gate: purple for CD90.1+; right box gate: blue for CD90.2+ CD3+CD8+ T cells. (C) Graphical quantitation of % CD3+CD8+CD90.2− and CD3+CD8+CD90.2+ as determined by flow cytometry. Group sizes were n = 4 for AAV2 and n = 5 for Cap-CD8. Error bars indicate mean ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Congenic BALB/c mice, CBy.PL(B6)-Thy1a/ScrJ, were used, in which their T cells express the CD90.1 (Thy1.1) surface antigen to track adoptively transferred Cap-CD8 generated in CD90.2 (Thy1.2) donor mice. (A) Gating strategy of antibody-labeled splenocytes stained with anti-mouse CD3-BV421, B220-BV605, CD8-A488, and CD90.2-APC. CD90.2+ CD8+ T cell gating was determined using a fluorescence minus one (-CD90.2-APC)-stained sample. (B) Representative dot plots of stained splenocytes from four BALB/c-CD90.1 (CD90.2-Cap-CD8) and BALB/c-CD90.2 control mice at 4 weeks following Cap-CD8 adoptive transfer. Left box gate: purple for CD90.1+; right box gate: blue for CD90.2+ CD3+CD8+ T cells. (C) Graphical quantitation of % CD3+CD8+CD90.2− and CD3+CD8+CD90.2+ as determined by flow cytometry. Group sizes were n = 4 for AAV2 and n = 5 for Cap-CD8. Error bars indicate mean ± SD. Rationally designed mutations of AAV2 with targeted substitutions of surface-exposed tyrosine residues with phenylalanine at positions 444, 500, and 730 [AAV2 (Y-F)] disrupted capsid polyubiquitination and shifted the trafficking of viral particles from the proteasome to the nucleus.28Zhong L. Li B. Mah C.S. Govindasamy L. Agbandje-McKenna M. Cooper M. Herzog R.W. Zolotukhin I. Warrington Jr., K.H. Weigel-Van Aken K.A. et al.Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses.Proc. Natl. Acad. Sci. USA. 2008; 105: 7827-7832Crossref PubMed Scopus (425) Google Scholar, 29Markusic D.M. Herzog R.W. Aslanidi G.V. Hoffman B.E. Li B. Li M. Jayandharan G.R. Ling C. Zolotukhin I. Ma W. et al.High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.Mol. Ther. 2010; 18: 2048-2056Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Together, these (Y-F) substitutions substantially improved vector transduction efficiency29Markusic D.M. Herzog R.W. Aslanidi G.V. Hoffman B.E. Li B. Li M. Jayandharan G.R. Ling C. Zolotukhin I. Ma W. et al.High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.Mol. Ther. 2010; 18: 2048-2056Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar and reduced capsid antigen processing and presentation on hepatocyte major histocompatibility complex (MHC) class I molecules.27Martino A.T. Basner-Tschakarjan E. Markusic D.M. Finn J.D. Hinderer C. Zhou S. Ostrov D.A. Srivastava A. Ertl H.C. Terhorst C. et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (119) Google Scholar We have described the design of an AAV2 capsid library incorporating two of the (Y-F) substitutions at positions 444 and 500, and following three rounds of in vivo selection on mouse liver identified two AAV2 variants, AAV2-LiA and AAV2-LiC, with equivalent transduction efficiency to AAV8.30Marsic D. Govindasamy L. Currlin S. Markusic D.M. Tseng Y.S. Herzog R.W. Agbandje-McKenna M. Zolotukhin S. Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.Mol. Ther. 2014; 22: 1900-1909Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Sequencing of the variants revealed that AAV2-LiA had 14 aa substitutions (including Y444F and Y500F), whereas AAV2-LiC had 4 aa substitutions (including Y500F) compared with wild-type AAV2. Because our previous study focused on AAV2 (Y-F) avoidance of Cap-CD8,27Martino A.T. Basner-Tschakarjan E. Markusic D.M. Finn J.D. Hinderer C. Zhou S. Ostrov D.A. Srivastava A. Ertl H.C. Terhorst C. et al.Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells.Blood. 2013; 121: 2224-2233Crossref PubMed Scopus (119) Google Scholar it was unclear what the relative contribution of each (Y-F) substitution had on capsid antigen presentation. Therefore, in addition to testing the relative capsid immunogenicity of two new variants with comparable transduction efficiency to AAV8, we were also able to address the requirement of (Y-F)-730 and multiple (Y-F) substitutions for attenuating capsid antigen presentation. To rule out the potential generation of new Ld-restricted CD8+ T cell epitopes for AAV2-LiA and AAV2-LiC in BALB/c mice (H-2d haplotype), we performed in silico prediction of MHC class I capsid epitopes. The VPQYGYLTL epitope was similarly ranked for wild AAV2, AAV2-LiA, and AAV2-LiC capsids, suggesting that this dominant epitope may be conserved in AAV2-LiA and AAV2-LiC (Table 1). An interferon gamma (IFNγ) ELISpot assay conducted on splenocytes collected from BALB/c mice receiving an intramuscular injection of either AAV2 (n = 2), AAV2-LiA (n = 3), or AAV2-LiC (n = 3) vectors all showed an increase in IFNγ spot-forming units following stimulation with VPQYGYLTL, demonstrating that this epitope is still processed and presented by both variants (Figure 4).Table 1Comparison of High to Low Binding H2Ld Predicted Epitopes Using In Silico PredictionAAV2AAV2-LiAAAV2-LiCStartEndPeptideStartEndPeptideStartEndPeptide534542FPQSGVLIF534542FPQSGVLIF534542FPQSGVLIF631639SPLMGGFGL631639SPLMGGFGL631639SPLMGGFGL363371PPFPADVFM363371PPFPADVFM363371PPFPADVFM372380VPQYGYLTLaThe dominant epitope used in this study is in bold. The new predicted epitope for AAV-LiC is underlined.372380VPQYGYLTLaThe dominant epitope used in this study is in bold. The new predicted epitope for AAV-LiC is underlined.372380VPQYGYLTLaThe dominant epitope used in this study is in bold. The new predicted epitope for AAV-LiC is underlined.307315RPKRLNFKL307315RPKRLNFKL502510WPGATTYHLaThe dominant epitope used in this study is in bold. The new predicted epitope for AAV-LiC is underlined.165173QPARKRLNF165173QPARKRLNF307315RPKRLNFKL621629IPHTDGHFH621629IPHTDGHFH165173QPARKRLNF437445LIDQYLYYL437445LIDQYLYFL621629IPHTDGHFH651659TPVPANPST651659TPVPANPST437445LIDQYLYYL724732RPIGTRYLT724732RPIGTRYLT651659TPVPANPST4553LVLPGYKYL4553LVLPGYKYL724732RPIGTRYLT653661VPANPSTTF653661VPANPSTTF4553LVLPGYKYL276284STPWGYFDF276284STPWGYFDF653661VPANPSTTF310318RLNFKLFNI310318RLNFKLFNI276284STPWGYFDF190198QPPAAPSGL190198QPPAAPSGL310318RLNFKLFNI515523SLVNPGPAM515523SLVNPGPAM190198QPPAAPSGL639647LKHPPPQIL639647LKHPPPQIL515523SLVNPGPAM407415NNFTFSYTF407415NNFTFSYTF639647LKHPPPQIL462470FSQAGASDI462470FSQAGASDI407415NNFTFSYTF357365AHQGCLPPF357365AHQGCLPPF462470FSQAGASDI346354SEYQLPYVL346354SEYQLPYVL357365AHQGCLPPF186194QPLGQPPAA186194QPLGQPPAA346354SEYQLPYVL403411LRTGNNFTF403411LRTGNNFTF186194QPLGQPPAA370378FMVPQYGYL370378FMVPQYGYL403411LRTGNNFTF502510WTGATKYHL502510WTGATKYHL370378FMVPQYGYL362370LPPFPADVF362370LPPFPADVF362370LPPFPADVF435443NPLIDQYLY435443NPLIDQYLY435443NPLIDQYLY508516YHLNGRDSL508516YHLNGRDSL508516YHLNGRDSL401409QMLRTGNNF401409QMLRTGNNF401409QMLRTGNNF715LPDWLEDTL715LPDWLEDTL715LPDWLEDTL298306RLINNNWGF298306RLINNNWGF298306RLINNNWGF629637HPSPLMGGF629637HPSPLMGGF629637HPSPLMGGF704712YNKSVNVDF704712YNKSVNVDF704712YNKSVNVDFa The dominant epitope used in this study is in bold. The new predicted epitope for AAV-LiC is underlined. Open table in a new tab AAV2-LiA and AAV2-LiC ApoE-hAAT-F9 vectors were evaluated at a dose of 1 × 1011 vg in the Cap-CD8 immune-competent mouse model. Mice transduced with either AAV2-LiA or AAV-LiC vectors expressed elevated levels of hFIX protein as compared with AAV2 (Figures 5A and 6A ). Yet, contrary to wild-type AAV2 (Figure 1B), no initial loss in hFIX expression was observed in Cap-CD8-treated mice compared with controls (Figures 5A and 6A). At study endpoint, a small but significant decrease in circulating hFIX levels was observed in AAV2-LiC-injected, but not AAV2-LiA-injected, animals. Plasma ALT levels were unchanged over time in AAV2-LiA mice receiving Cap-CD8 (Figure 5B), whereas a rise in ALT levels (∼5-fold over normal levels of 9.3 ± 4.3 U/L) was detected in two of five AAV2-LiC mice receiving Cap-CD8 at 4 weeks (Figure 6B). Liver tissue from each group was collected at 1 and 4 weeks post vector administration, and immunostaining for hFIX (red) and CD90.2+ T cells (green) was performed on liver sections from control and Cap-CD8 mice. Representative sections at 40× magnification from each vector (AAV2-LiA and AAV2-LiC) and group at 1 and 4 weeks are provided in Figures 5C and 6C, respectively. Despite a moderate decrease in circulating hFIX protein detected in AAV2-LiC Cap-CD8 mice, quantitation of hFIX+ hepatocytes at 4 weeks did not reveal a significant decrease in hFIX+ hepatocytes in Cap-CD8 compared with control mice in both AAV2-LiA- and AAV2-LiC-treated mice (Figures 5D and 6D). As with AAV2-injected mice, no CD90.2+ Cap-CD8 cell infiltration was observed in liver sections, and flow cytometry staining of splenocytes at 4 weeks did not detect CD90.2+ Cap-CD8 in either AAV2-LiA or AAV2-LiC Cap-CD8 mice (data not shown).Figure 6AAV2-LiC Mutant Capsid Has a D
Referência(s)