Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare? (2020)

Recombinant AAV (rAAV) vectors are a key component of an emergent therapeutic paradigm with a demonstrated definitive benefit for genetic diseases. Immune responses are among the most challenging barriers to human gene therapy. Viral vectors are highly advantageous because of their evolved ability to negotiate complex intracellular pathways to efficiently deliver a DNA payload; however, the human immune system has evolved multiple pathways, including innate pathogen-associated molecular pattern (PAMP) sensors that trigger adaptive effector functions to eliminate infected cells.1Newton K. Dixit V.M. Signaling in innate immunity and inflammation.Cold Spring Harb. Perspect. Biol. 2012; 4: a006049Crossref PubMed Scopus (1019) Google Scholar While rAAV product developers cannot avoid using AAV capsid, which is the source of viral peptides that render transduced cells targets for capsid-specific cytotoxic T lymphocytes (CTLs), the elimination of immune co-stimulatory features is important. Unmethylated CpG dinucleotide-based motifs (CpGs) are known PAMPs that bind and dimerize monomeric TLR9 expressed in human dendritic cells2Hartmann G. Weiner G.J. Krieg A.M. CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells.Proc. Natl. Acad. Sci. USA. 1999; 96: 9305-9310Crossref PubMed Scopus (559) Google Scholar,3Ohto U. Ishida H. Shibata T. Sato R. Miyake K. Shimizu T. Toll-like receptor 9 contains two DNA binding sites that function cooperatively to promote receptor dimerization and activation.Immunity. 2018; 48: 649-658.e4Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and have been shown to cause activation of the Toll-like receptor (TLR)9-MyD88 signaling pathway, thereby promoting CTL responses to AAV vectors in non-clinical models.4Zhu J. Huang X. Yang Y. The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice.J. Clin. Invest. 2009; 119: 2388-2398Crossref PubMed Scopus (207) Google Scholar, 5Shirley J.L. Keeler G.D. Sherman A. Zolotukhin I. Markusic D.M. Hoffman B.E. Morel L.M. Wallet M.A. Terhorst C. Herzog R.W. Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T Cells.Mol. Ther. 2019; 28 (this issue): 758-770Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Xiang Z. Kurupati R.K. Li Y. Kuranda K. Zhou X. Mingozzi F. High K.A. Ertl H.C.J. The effect of CpG sequences on capsid-specific CD8+ cell responses to AAV vector gene therapy.Mol. Ther. 2019; 28 (this issue): 771-783Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Polynucleotides containing unmethylated CpGs are adjuvants used in vaccine development to stimulate strong cellular immune responses.7Bode C. Zhao G. Steinhagen F. Kinjo T. Klinman D.M. CpG DNA as a vaccine adjuvant.Expert Rev. Vaccines. 2011; 10: 499-511Crossref PubMed Scopus (557) Google Scholar Details now available for eight hemophilia B gene therapy trials that used differing codon-modification strategies resulting in a broad range of CpG content (0- to 5-fold of wild type) in the factor IX (FIX) open reading frame (ORF) reveal that low CpG correlates strongly to long-term expression. Herein is provided a perspective that unmethylated CpG content in AAV vectors is the “key” attribute that triggers transgene expression-limiting immune responses in humans and that novel clinical vector production strategies to increase CpG methylation should be developed. A discussion of the role of CpGs in AAV vectors and their contribution to immunotoxicity and loss of transgene expression in hemophilia gene therapy was catalyzed by the report by Chapin and colleagues (J. Chapin et al., 2018, 14th Workshop on Novel Technologies and Gene Therapies for Hemophilia, conference), which reported unexpected loss of FIX expression in 7 of 8 patients in their clinical trial using AAV8-FIXsc (Padua) investigational product BAX335. They hypothesized that CpG enrichment resulting from “codon-optimization” of the FIX ORF was the root cause of the CTL formation that eliminated transduced cells. This hypothesis is supported by results from seven other AAV-based gene therapy trials for hemophilia B reporting long-term follow-up, as summarized in Table 1. Among the variables, including serotype, expression cassette configuration, production method, vector genome (vg) and estimated total capsid dose, and the use of immune-suppression, low CpG content is the only parameter that fully correlates with long-term FIX expression. Codon modification was used to remove the 19 CpGs present in wild-type FIX cDNA in all four trials that reported durable FIX expression in all (33 combined) subjects, as well as the absence of or modest CTL responses that were easily controlled by transient immune suppression. In contrast, a different codon modification approach that aimed to increase the translational kinetics of the expression cassette and, in the process, increasing CpGs by approximately 5-fold over wild-type cDNA, was used in the three trials that reported stronger CTL responses that were not well-controlled by immune suppression. In the two studies that published outcomes, loss of FIX expression in all but one of 14 subjects was reported. These data are consistent with unmethylated CpGs in AAV vectors as the primary trigger for efficacy-limiting CTL responses in humans. Higher doses that render more transduced cells targets for capsid-specific CTLs and AAV serotypes that are more efficiently taken up by TLR9-expressing dendritic cells are likely important contributing factors.Table 1AAV Gene Therapy Clinical Trials for Hemophilia BSponsorSerotype/ConfigurationaGenome configuration: ss, single-stranded genome; sc, self-complementary genome.No. of CpG in ORFProductionDose (×1012)ImmunologyOutcomes(vg/kg)(∼cp/kg)ISbImmune suppression: –, not used; +, minority of subjects; ++, majority of subjects.CTLcCapsid-specific CTLs by IFN-γ ELISPOT: +, minority of subjects; ++, majority of subjects.Peak FIXDurationCHOP, StanfordAvigenAAV2-FIX/ss19dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman Lecture (WT)HEK22–++12% (n = 1)<3 monthsUCL, St JudeAAV8-FIX/sc0dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman LectureHEK0.2–21–10++2%–11% (n = 10)>1 yearShire (BAX335)AAV8-FIX Padua/sc99dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman LectureHEK0.2–3ND++++4%–45% (n = 8)<3 monthsCHOPAAV8-FIX19/ss94eHigh and Anguela, 2016, USTPO 20160375110ND1–2ND++++eHigh and Anguela, 2016, USTPO 20160375110NDNDPfizer (SPK-9001)AAVSPK-FIX Padua/ss0dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman LectureHEK0.51.5–2.5++34% (n = 10)>1 yearUniqure (AMT060)AAV5-FIX/sc0dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman LectureBac2040++7% (n = 5)>1 yearDimension (DTX101)AAVrh10-FIX/ss96dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman LectureHEK1.6–5ND++++3%–8% (n = 6)<3 monthsUniqure (AMT061)AAV5-FIX Padua/sc0dNathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman LectureBac2040–+47% (n = 3)>1 yeara Genome configuration: ss, single-stranded genome; sc, self-complementary genome.b Immune suppression: –, not used; +, minority of subjects; ++, majority of subjects.c Capsid-specific CTLs by IFN-γ ELISPOT: +, minority of subjects; ++, majority of subjects.d Nathwani, 2019, American Society for Hematology Annual Meeting, Ham Wasserman Lecturee High and Anguela, 2016, USTPO 20160375110 Open table in a new tab The term “codon optimization” has been used to describe various codon modifications in rAAV vectors, often without clear definition. Given that (1) the number of DNA sequences that can encode a single protein is large, (2) the poor clinical outcomes associated with codon modifications that increase CpG content as shown in Table 1, and (3) the likely exclusion of benefit from future AAV products due to seroconversion8Kruzik A. Fetahagic D. Hartlieb B. Dorn S. Koppensteiner H. Horling F.M. Scheiflinger F. Reipert B.M. de la Rosa M. Prevalence of anti-adeno-associated virus immune responses in international cohorts of healthy donors.Mol. Ther. Methods Clin. Dev. 2019; 14: 126-133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar for human subjects that receive vectors that fail to achieve durable therapeutic effect, it is clear that guidelines for codon modification of rAAV vectors for in vivo gene therapy should be developed and shared. The algorithms previously used to increase the efficiency of recombinant human protein expression in heterologous production cells that increase CpG content9Gustafsson C. Govindarajan S. Minshull J. Codon bias and heterologous protein expression.Trends Biotechnol. 2004; 22: 346-353Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar,10Fath S. Bauer A.P. Liss M. Spriestersbach A. Maertens B. Hahn P. Ludwig C. Schafer F. Graf M. Wagner R. Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.PLos One. 2011; 6: e17596Crossref PubMed Scopus (118) Google Scholar should not be used. Clinical experience for hemophilia B supports the concept that codon modification to remove CpGs is beneficial and should be used for other rAAV investigational products where the route of administration is immunologically responsive. The sharing of CpG content in rAAV expression cassettes by sponsors developing gene therapies for other diseases (e.g., hemophilia A and DMD) would further help define best practices for the field and optimal benefit for human subjects. Since safety and long-term expression are the key attributes for most AAV gene therapies, a strategy of “hasten slowly” for codon modification is prudent—prioritize first and foremost the elimination and avoidance of PAMPs. Two additional lines of evidence support the idea that hypomethylation is an important product attribute flaw in the current generation of AAV vectors. The first is that efficacy-limiting CTLs and loss of expression were observed in the first liver-directed AAV-FIX clinical trial (CHOP, Avigen)11Manno 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 (1568) Google Scholar in which the FIX cDNA (i.e., with wild-type CpG content) was used, albeit in the absence of immune suppression regimens that were subsequently developed. The second line of evidence stems from direct biochemical analysis of marked CpG hypomethylation in AAV generated using transient transfection12Tóth R. Mészáros I. Hüser D. Forró B. Marton S. Olasz F. Bányai K. Heilbronn R. Zádori Z. Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).Viruses. 2019; 11: 38Crossref Scopus (10) Google Scholar. Maneuvers used in the clinic to mitigate this AAV vector product immunostimulatory feature—namely reduction of CpGs by codon modification—and immune suppression of subjects until capsid peptides have cleared from transduced tissues have not always prevented CTL generation and loss of transgene expression. A novel approach that takes more direct aim at the root cause of hypomethylation in AAV vectors is to increase CpG methylation by development of improved production technologies. While such strategies would vary depending on production cell line type (e.g., mammalian or insect) and mode of introduction (e.g., transfection or infection) of the genes required for vector generation, the main objective would be to provide enough targeted methyl transferase during production of input vector DNA (e.g., plasmid) and during vector genome replication in production cells to achieve ∼75% CpG methylation, comparable to the level in human DNA. To meet this goal, accurate understanding of the provenance of AAV expression cassette DNA is required. For example, in the generation of AAV2 vectors by transient transfection with plasmid DNA in HEK293 cells, a fraction of packaged genomes was reported to be of plasmid DNA origin, i.e., rescued directly from vector plasmid.13Hauck B. Mingozzi F. Arruda V. High K.A. Wright J.F. Investigation of biochemical factors that may influence immunogenicity of AAV2 vectors.Mol. Ther. 2006; 13: S45Abstract Full Text PDF Google Scholar A model for AAV expression cassette rescue and packaging from plasmid DNA showing pathways for genomes excised from plasmid (left) and canonical14Ward P. Elias P. Linden R.M. Rescue of the adeno-associated virus genome from a plasmid vector: evidence for rescue by replication.J. Virol. 2003; 77: 11480-11490Crossref PubMed Scopus (19) Google Scholar replication-derived AAV genomes (right) is shown in Figure 1. The prokaryotic DNA genomes are expected to contain only unmethylated CpGs, while the genomes replicated in the mammalian cell would contain some methylated CpGs, though few because of insufficient methyl transferase available during the rapid kinetics of replication and packaging.12Tóth R. Mészáros I. Hüser D. Forró B. Marton S. Olasz F. Bányai K. Heilbronn R. Zádori Z. Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).Viruses. 2019; 11: 38Crossref Scopus (10) Google Scholar Calculation shows that, for typical transient transfection methods, vector plasmid copy number input is comparable to the number of AAV packaged genomes produced, supporting that both pathways shown in Figure 1 should be considered in the development of strategies to increase CpG methylation. In reference to Figure 1, CpG methylation of the vector plasmid (a) and during AAV genome replication (b) represent strategies for methyl transferase supplementation. Similar considerations can be used for other AAV vector production systems, for example, to address CpG methylation limitations in Holometabola.15Provataris P. Meusemann K. Niehuis O. Grath S. Misof B. Signatures of DNA methylation across insects suggest reduced DNA methylation levels in holometabola.Genome Biol. Evol. 2018; 10: 1185-1197Crossref PubMed Scopus (48) Google Scholar The frequency of unmethylated CpG motifs in the genomes of AAV vectors prepared for human gene therapy is likely a critical quality attribute for vectors intended for in vivo administration, and specifications to ensure adequate innate immune histocompatibility should be established. Type I IFN Sensing by cDCs and CD4+ T Cell Help Are Both Requisite for Cross-Priming of AAV Capsid-Specific CD8+ T CellsShirley et al.Molecular TherapyNovember 14, 2019In BriefImmune responses complicate the use of adeno-associated virus (AAV) vectors in human gene therapy. Shirley et al. define a mechanism by which cross-presentation of viral capsid results in activation of CD8+ T cells, which involves sensing of IFN I by conventional dendritic cells and co-stimulation by CD4+ T helper cells. Full-Text PDF Open ArchiveThe Effect of CpG Sequences on Capsid-Specific CD8+ T Cell Responses to AAV Vector Gene TransferXiang et al.Molecular TherapyNovember 20, 2019In BriefStimulation of naive, but not memory CD8+ T cells, to capsids of AAV vectors depends on CpG motifs within the vectors’ genomes. Naive T cells respond to CpGhi but not CpGlow or empty vectors. Memory T cells respond vigorously to CpGlow or empty vectors, but not to CpGhigh vectors. Full-Text PDF Open Archive