[This corrects the article DOI: 10.1016/j.omtm.2023.101150.].

Strategies to administer AAV vectors safely and effectively to subjects with pre-existing antibodies are needed because of the high prevalence of wild-type AAV infection and increasing use of AAV-based products. Capsid antibodies block transduction, are cross-reactive between serotypes, 1 Kruzik 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: 126-133 Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar and recipients of AAV vectors generate high-titer antibodies that are durable for years. 2 George L.A. Ragni M.V. Rasko J.E.J. Raffini L.J. Samelson-Jones B.J. Ozelo M. Hazbon M. Runowski A.R. Wellman J.A. Wachtel K. et al. Long-term follow-up of the first in human intravascular delivery of AAV for gene transfer: AAV2-hFIX16 for severe hemophilia B. Mol. Ther. 2020; 28: 2073-2082 Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Immune complexes formed by antibody binding to AAV have been implicated in activation of the complement cascade 3 Zaiss A.K. Cotter M.J. White L.R. Clark S.A. Wong N.C.W. Holers V.M. Bartlett J.S. Muruve D.A. Complement is an essential component of the immune response to adeno-associated virus vectors. J. Virol. 2008; 82: 2727-2740 Crossref PubMed Scopus (107) Google Scholar associated with serious adverse events in high-dose clinical trials. Strategies to overcome this problem include discovery or creation of novel, immunologically distinct capsids 4 Bartel M. Schaffer D. Büning H. Enhancing the clinical potential of AAV vectors by capsid engineering to evade pre-existing immunity. Front. Microbiol. 2011; 2: 204https://doi.org/10.3389/fmicb.2011.00204 Crossref PubMed Scopus (82) Google Scholar that have the potential to increase the number of subjects eligible for a first-time treatment but will themselves induce high-titer antibodies that preclude AAV vector re-administration. Removal of AAV antibodies in subjects before vector administration by plasmapheresis, in vivo enzymatic digestion of total IgG, and prevention of antibody formation by immune suppression are promising approaches 5 Schulz M. Levy D.I. Petropoulos C.J. Bashirians G. Winburn I. Mahn M. Somanathan S. Cheng S.H. Byrne B.J. Binding and neutralizing anti-AAV antibodies: detection and implications for rAAV-mediated gene therapy. Mol. Ther. 2023; 31: 616-630 Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar but may not achieve sufficient antibody reduction and pose additional risks. Solving the antibody problem is key to maximize AAV gene therapy safety, efficacy, and wide utilization, including enablement of re-dosing to maintain the benefit achieved after an initial systemic dose that delivers therapeutic genes to liver or neuro-muscular cells but that fades with time or pediatric patient growth.

[This corrects the article DOI: 10.1016/j.omtm.2023.02.008.].

Background aims: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost. Methods: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Results: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000-750 000 vg/cell AAV and 0.8-4 mg sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 mg sgRNA. Conclusions: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics. (c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

The pace and diversity of adeno-associated virus (AAV) vector based clinical trials over the last 5 years has been high, with definitive therapeutic benefits observed with the two products licensed by the US Food and Drug Administration, as well as several investigational products in pivotal trials demonstrating the paradigm-shifting potential of AAV based gene therapy for serious unmet medical needs. However, this excitement has been tempered by the emergence of serious adverse events (SAEs) in several clinical trials, many of which seem to be caused by innate and adaptive immune responses to the vector (https://www.fda.gov/media/151599/download).1Mullard A. Gene therapy community grapples with toxicity issues, as pipeline matures.Nat. Drug Discov. 2021; 20: 804-805https://doi.org/10.1038/d41573-021-00164-xCrossref PubMed Scopus (36) Google Scholar Product critical quality attributes (CQAs) are by definition features that must be within an appropriate limit, range, or distribution to ensure product safety and efficacy. The latter are also inextricably linked to manufacturing processes—the product is the process—and efforts should focus on understanding the product features that have contributed to vector-related SAEs and how these can be influenced by manufacturing process design. The challenges of comparability studies to support process evolution during clinical development notwithstanding, establishing high capacity, cost-effective manufacturing processes in the absence of a comprehensive understanding of product CQAs is putting the cart before the horse. Defining all product safety CQAs, including those not identified in pre-clinical studies but that subsequently emerge from clinical experience, is a pre-requisite to finalizing a manufacturing process. Ease of scale-up and cost efficiency are important co-requisites that will ultimately be required for the industrialization of AAV gene therapy. In contrast with recombinant therapeutic human proteins made in mammalian cell lines, the non-self-nature of AAV vectors with respect to the human immune system is more akin to vaccines designed to stimulate immune responses. The presence of viral antigens and pathogen-associated molecular patterns (PAMPs) contributed by AAV capsid amino acid sequences, residual viral DNA sequences, microbial epigenetic PAMPs in vector genomes,2Rumachik N.G. Malaker S.A. Poweleit N. Maynard L.H. Adams C.M. Leib R.D. Cirolia G. Thomas D. Stamnes S. Holt K. et al.Methods matter: standard production platforms for recombinant AAV produce chemically and functionally distinct vectors.Mol. Ther. Meth. Clin. Dev. 2020; 18: 98-118https://doi.org/10.1016/j.omtm.2020.05.018Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar and the use of microbial components (recombinant plasmids and viruses) for AAV manufacture together ensure that the in vivo administration of vectors will trigger innate and adaptive immune responses. Achieving safety and durable efficacy may require significant vector and manufacturing process design changes in response to clinical experience. Byrne and colleagues3Byrne B.J. Corti M. Muntoni F. Considerations for systemic use of gene therapy.Mol. Ther. 2021; 29: 422-423https://doi.org/10.1016/j.ymthe.2021.01.016Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar recently compared the immunological challenges using the systemic administration of AAV vectors with those encountered during the development of organ transplantation to support that improved immune management is key to mitigate immunotoxicity after administration—transplantation—of AAV vectors to human subjects. Building on this comparison, the establishment of tissue typing to maximize histocompatibility, i.e., minimize the immunological differences between transplanted tissue and recipient is another key requirement for successful organ transplantation. By analogy, identifying and decreasing potentially inflammatory vector attributes by manufacturing process design improvements is important to complement better immune management. One prudent objective in response to the emergence of immune response-related SAEs is to critically evaluate, and where necessary, modify upstream (cell culture-based vector generation) and downstream (vector purification) manufacturing processes to decrease product immunogenicity. As has been recognized and highlighted by other investigators, there remains a need to establish AAV upstream processes that can meet projected vector production capacity as clinical programs progress and more products are commercialized.4Escandell J.M. Pais D.A.M. Carvalho S.B. Vincent K. Gomes-Alves P. Alves P.M. Leveraging rAAV bioprocess understanding and next generation bioanalytics development.Curr. Opin. Biotech. 2022; 74: 271-277https://doi.org/10.1016/j.copbio.2021.12.009Crossref PubMed Scopus (5) Google Scholar To highlight the manufacturing capacity gap and potential for improvement, a back-of-the-envelope calculation estimates that approximately 1019 AAV vectors have been manufactured in total for administration to human subjects in clinical trials and commercial dosing to date, corresponding with approximately 100 g recombinant AAV (rAAV), a quantity that for a typical monoclonal antibody can be manufactured from 20 L of cell culture. The current generation of rAAV production technologies using microbial helper components has supported remarkable progress, but the low volumetric production efficiency combined with product-associated immunotoxicities revealed by clinical experience argue that the acceleration of alternative AAV production platforms should be an important future direction. Engineered cell lines that can be induced to express the gene(s) required for product generation represent the industry standard for recombinant therapeutic proteins such as monoclonal antibodies. The challenges that have hindered the development of such cell lines for AAV vector production, including the complexity and cytostatic/cytotoxic nature of the multiple gene products required, are being overcome by advancing technologies, including more efficient and better choreographed induction of production gene expression. Importantly, the continued development of such producer cell lines should help to decrease the immunogenicity of the current generation of AAV vectors, e.g., by eliminating the need for microbial helper components, increasing vector genome packaging efficiency to decrease the fraction of empty capsids in the upstream harvest, and decreasing unmethylated CpG motifs in AAV vector genomes implicated in TLR9 innate signaling. Accelerating development of such producer cell lines could meet the twin aims of better process scalability and decreased product immunogenicity. For vector purification, while standard bioprocess steps developed for protein purification have been successfully adapted for AAV vectors, one unique and challenging problem is the heterogeneity of AAV particles generated using current upstream platforms. There is variability in the nomenclature used to describe the diverse AAV particles formed. Table 1 proposes division of the AAV particle types into four categories and summarizes their associated immunological risks. The term "partials" is proposed to be reserved for AAV particles that contain a portion of the intended vector genome, which may be common for oversized vector genomes, that contribute to target cell transduction.5Sihn C.-R. Handyside B. Liu S. Zhang L. Murphy R. Yates B. Xie L. Torres R. Russell C.B. O'Neill C.A. et al.Molecular analysis of AAV5-hFVIII-SQ vector-genome-processing kinetics in transduced mouse and nonhuman primate livers.Mol. Ther. Meth. Clin. Dev. 2022; 24: 142-153https://doi.org/10.1016/j.omtm.2021.12.004Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Empty capsids are generally the most abundant particle type generated in cell culture production because of the inefficiency of vector genome packaging into pre-formed capsids and they are difficult to separate from the target vector. The most efficient method to remove empty capsids that is used by some sponsors is a gradient ultracentrifugation step. Column chromatography can also decrease empty capsids, but with a lower resolution and less robustly than ultracentrifugation. Some sponsors have chosen to accept the co-purification of empty capsids with the vector product because the steps for their removal are difficult to scale up and validate; such purified products may contain 50%–90% empty capsids, translating to a 2- to 10-fold higher capsid antigen dose for a given vector genome dose. Therefore, the decision of whether or not to remove empty capsids can represent a choice between greater process scalability versus lower product immunogenicity; which should prevail? The emergence of serious immunotoxicities after high-dose AAV vector administration, including complement activation that are likely capsid dose dependent, argues that total capsid dose is a critical quality attribute for safety at high vector doses and that empty capsids should be efficiently removed.Table 1Proposed categorization of AAV particles generated during cell cultureAAV particle descriptionOther namesCategoryImmunological risk1.Packaged full-length vector genomes"Fulls"ProductInherent product risk2.Packaged partial vector genomes"Partials"Partial productReduced transduction efficiency leads to higher dose requirement3.Packaged DNA impurities (nuclease-resistant cell and helper DNA)Residual packaged DNA impuritiesProduct-related impuritiesMicrobial DNA PAMPs and ORFs lead to innate and adaptive immune responses4.Empty capsids"Empties"Product-related impurityLead to multi-fold increase in total viral capsid dose Open table in a new tab

Hemophilia is a target for adeno-associated virus (AAV) gene therapy, but factors contributing to efficacy-limiting immune responses must be better understood and addressed. Cytotoxic T Lymphocyte (CTL) responses to AAV destroy transduced cells displaying AAV antigens and eliminate therapeutic factor IX expression. The CTL response is initiated when Toll-Like Receptor 9 (TLR9) encounters unmethylated CpG dinucleotide pathogen associated molecular patterns in AAV genomes. AAV vector genomes are comprised of a promoter-driven transgene flanked by Inverted Terminal Repeat (ITR) sequences. ITRs are the only required viral sequences in AAV vectors, and are usually derived from AAV2. We hypothesize that ITRs with lower CpG frequency will initiate less signaling through TLR9. We compared ITR sequences from AAV serotypes 1-8, avian, bat and bovine AAV, and a CpG-freeITR in HEKTLR9 reporter cells. Six ITR sequences, including AAV2, have 16 CpGs; AAV1 and AAV3 have 14 CpGs; AAV8 has 12 and AAV4 has 10. The Bat ITR has only 4 CpGs. We found significant differences in TLR9 activation after ITR exposure in HEKTLR9. AAV3ITR was the strongest TLR9 activator, with 200% the signal of AAV2ITR. In contrast, AAV8ITR was least stimulatory, activating TLR9 48% less than AAV2ITR and insignificantly less than the CpG freeITR. Non-denaturing agarose electrophoresis showed banding patterns consistent with base paired ITR secondary structures, which migrate faster than polyT unpaired controls. We also tested if methylation of CpGs would decrease TLR9 activation and observed a decrease in TLR9 activation to levels equal to CpG-freeITR. Migration of methylated ITRs was indistinguishable from unmethylated counterparts after electrophoresis, suggesting no change in secondary structure. This study demonstrates the potential for decreasing AAV immunogenicity through alternative ITR design. Hemophilia is a target for adeno-associated virus (AAV) gene therapy, but factors contributing to efficacy-limiting immune responses must be better understood and addressed. Cytotoxic T Lymphocyte (CTL) responses to AAV destroy transduced cells displaying AAV antigens and eliminate therapeutic factor IX expression. The CTL response is initiated when Toll-Like Receptor 9 (TLR9) encounters unmethylated CpG dinucleotide pathogen associated molecular patterns in AAV genomes. AAV vector genomes are comprised of a promoter-driven transgene flanked by Inverted Terminal Repeat (ITR) sequences. ITRs are the only required viral sequences in AAV vectors, and are usually derived from AAV2. We hypothesize that ITRs with lower CpG frequency will initiate less signaling through TLR9. We compared ITR sequences from AAV serotypes 1-8, avian, bat and bovine AAV, and a CpG-freeITR in HEKTLR9 reporter cells. Six ITR sequences, including AAV2, have 16 CpGs; AAV1 and AAV3 have 14 CpGs; AAV8 has 12 and AAV4 has 10. The Bat ITR has only 4 CpGs. We found significant differences in TLR9 activation after ITR exposure in HEKTLR9. AAV3ITR was the strongest TLR9 activator, with 200% the signal of AAV2ITR. In contrast, AAV8ITR was least stimulatory, activating TLR9 48% less than AAV2ITR and insignificantly less than the CpG freeITR. Non-denaturing agarose electrophoresis showed banding patterns consistent with base paired ITR secondary structures, which migrate faster than polyT unpaired controls. We also tested if methylation of CpGs would decrease TLR9 activation and observed a decrease in TLR9 activation to levels equal to CpG-freeITR. Migration of methylated ITRs was indistinguishable from unmethylated counterparts after electrophoresis, suggesting no change in secondary structure. This study demonstrates the potential for decreasing AAV immunogenicity through alternative ITR design.

Host immune responses that limit durable therapeutic gene expression and cause clinically significant inflammation remain a major barrier to broadly successful development of adeno-associated virus (AAV)-based human gene therapies. In this article, mechanisms of humoral and cellular immune responses to the viral vector are discussed. A perspective is provided that removal of pathogen-associated molecular patterns in AAV vector genomes to prevent the generation of innate immune danger signals following administration is a key strategy to overcome immunological barriers.

Preclinical efficacy and safety data in mice provide support for ex vivo β-globin gene correction to treat patients with sickle cell disease.

Gene therapy is at the forefront of the drive to bring the potential of cure to patients with genetic diseases. Multiple mechanisms of effective and efficient gene therapy delivery (eg, lentiviral, adeno-associated) for transgene expression as well as gene editing have been explored to improve vector and construct attributes and achieve therapeutic success. Recent clinical research has focused on recombinant adeno-associated viral (rAAV) vectors as a preferred method owing to their naturally occurring vector biology characteristics, such as serotypes with specific tissue tropisms, facilitated in vivo delivery, and stable physicochemical properties. For those living with hereditary diseases like hemophilia, this potential curative approach is balanced against the need to provide safe, predictable, effective, and durable factor expression. While in vivo studies of rAAV gene therapy have demonstrated amelioration of the bleeding phenotype in adults, long-term safety and effectiveness remain to be established. This review discusses vector biology in the context of rAAV-based liver-directed gene therapy for hemophilia and provides an overview of the types of viral vectors and vector components that are under investigation, as well as an assessment of the challenges associated with gene therapy delivery and durability of expression.

Adeno-associated virus (AAV) vector gene therapy is a promising treatment for a variety of genetic diseases, including hemophilia. Systemic administration of AAV vectors is associated with a cytotoxic immune response triggered against AAV capsid proteins, which if untreated can result in loss of transgene expression. Immunosuppression (IS) with corticosteroids has limited transgene loss in some AAV gene therapy clinical trials, but was insufficient to prevent loss in other studies. We used a nonhuman primate model to evaluate intensive T cell-directed IS combined with AAV-mediated transfer of the human factor IX (FIX) gene. Early administration of rabbit anti-thymocyte globulin (ATG) concomitant with AAV administration resulted in the development of anti-FIX antibodies, whereas delayed ATG by 5 weeks administration did not. The anti-FIX immune response was associated with increases in inflammatory cytokines, as well as a skewed Th17/regulatory T cell (Treg) ratio. We conclude that the timing of T cell-directed IS is critical in determining transgene-product immunogenicity or tolerance. These data have implications for systemically administered AAV gene therapy being evaluated for hemophilia A and B, as well as other genetic diseases.

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

Recombinant Adeno-Associated Virus (rAAV) are widely used for human gene therapy, now the basis for two licensed products (for RPE65-/-, and SMA) and numerous other investigational treatments for a range of diseases including hemophilia, cystic fibrosis, muscular dystrophy & many neurological disorders. Process development is complex, time consuming & expensive, with scale-up being a major technology challenge for commercialization. While progress has been made to implement clinical vector production (upstream) and purification (downstream) processes at large scale, required for high dose indications, many challenges remain in vector design, manufacturing methodology & product characterization. Ongoing innovation is required to understand the critical quality attributes of AAV vectors for human use, & to establish suitable and standardized analytical systems to monitor and control clinical manufacturing processes & characterize the purified vector products. 'Research-grade' vectors are not subject to the same control criteria; however, similar approaches are utilized on a routine basis in cores and research labs to manufacture viral vectors for discovery, investigational product development & IND-supporting studies. In that regard, academic centers should promote and implement good practices and make transferable protocols to accelerate gene therapy drug development. At the GVVC a high-throughput platform has been optimized to produce custom rAAVs with a rapid turnaround and rigorous analytical methods for quality control. These include qualified qPCR titer methodology, purity using a range of tests & functional activity (infectivity) verificdation of the recombinant virus. Furthermore, this core facility borrows from GXP practice for carefully segregated workflow to prevent product cross-contamination in a high throughput environment. the GVVC facility has manufactured over 5000 custom AAV vector batches for Stanford researchers & neuroscientists world-wide.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors has demonstrated definitive benefits for genetic diseases and has enormous future potential, representing an important part of the next paradigm of human therapeutics. However, many clinical trials with rAAV have reported varying degrees of immunotoxicity, potentially including that associated with the recently reported deaths of two subjects in a clinical trial for X-linked myotubular myopathy. Analogous to the identification and removal of immunogenic features of early-era monoclonal antibodies, thereby “humanizing” those products, while recognizing the immutable viral nature of the vector capsid, a similar strategy of humanizing addressable features of AAV vectors during their design is an opportunity to accelerate successful clinical product development. The synergistic nature of the multiple pathways that comprise human innate and adaptive immune responses combined with the consequences of failure to adequately control them after AAV-mediated gene delivery, including immunotoxicity, potential loss of transgene expression, and AAV antibody seroconversion preventing re-administration, support the need to identify and remove “microbial legacy” immunostimulatory features such as pathogen-associated molecular patterns (PAMPs).1Barton G.M. Kagan J.C. A cell biological view of Toll-like receptor function: regulation through compartmentalization.Nat. Rev. Immunol. 2009; 9: 535-542Crossref PubMed Scopus (522) Google Scholar This commentary focuses on one such PAMP, the unmethylated CpG motifs (PAMP CpG) commonly found in AAV vectors due to hypomethylation of vector genomes during their production2Tóth 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 (AA2).Viruses. 2019; 11: 38Crossref Scopus (10) Google Scholar and presence of expression cassette elements of microbial origin that are rich in CpGs. Herein we describe approaches to quantify the Toll-like receptor 9 (TLR9) innate immune pathway activation risk for DNA sequences of interest, e.g., rAAV expression cassettes under consideration as investigational products based on CpG/motif content and methylation, providing a tool to assess and guide reduction of TLR9-associated immunogenicity. PAMP CpG binds and dimerizes TLR9 molecules3Ohto U. Shibata T. Tanji H. Ishida H. Krayukhina E. Uchiyama S. Miyake K. Shimizu T. Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9.Nature. 2015; 520: 702-705Crossref PubMed Scopus (237) Google Scholar expressed in plasmacytoid dendritic cells (pDCs), leading via MyD88 to cellular immune responses.4Hartmann 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 An established and growing body of non-clinical5Zhu 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, 6Faust S.M. Bell P. Cutler B.J. Ashley S.N. Zhu Y. Rabinowitz J.E. Wilson J.M. CpG-depleted adeno-associated virus vectors evade immune detection.J. Clin. Invest. 2013; 123: 2994-3001Crossref PubMed Scopus (145) Google Scholar, 7Shirley 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 1 IFN sensing by cDCs and CD4+ T cell help are both requisite for cross-priming of AAV capsid-specific CD8+ cells.Mol. Ther. 2020; 28: 758-770Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Xiang 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. 2020; 28: 771-783Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar and clinical9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar evidence unsurprisingly supports the model that a viral capsid containing a genome with PAMP CpG stimulates innate and adaptive immune pathways, leading to the formation of capsid-specific cytotoxic T lymphoctes (CTLs). Figure 1 illustrates how high PAMP CpG levels in the expression cassette of an AAV vector lead to CTLs that eliminate transduced hepatocytes (Figure 1A), while a vector genome with a sub-threshold PAMP CpG level does not activate the TLR9-MyD88 pathway and spares transduced cells, leading to durable transgene expression (Figure 1B). Factors not shown in Figure 1 certainly contribute to these pathways, e.g., higher vector doses would be expected to increase the severity of the hepato-immunotoxicity shown in Figure 1A. Transient immune suppression is frequently used and partially effective in managing CTL responses in rAAV clinical studies but adds complexity and risk.10Samelson-Jones B.J. Finn J.D. Favaro P. Wright J.F. Arruda V.R. Timing of intensive immunosuppression impacts risk of transgene antibodies after AAV gene therapy in nonhuman primates.Mol. Ther. Methods Clin. Dev. 2020; 17: 1129-1138Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Avoidance of TLR9 activation by reducing PAMP CpG in AAV vector genomes during investigational product design is a promising approach to directly address the root cause. With recognition of the innate immunogenic risk of PAMP CpG in rAAV, vector design strategies, including codon modification of open reading frames and sequence changes in non-coding elements to reduce CpG dinucleotides, are becoming best practices. Complete CpG removal from an expression cassette is possible but has the potential to cause transprotein misfolding due to non-wild-type translational kinetics11Mauro V.P. Chappell S.A. A critical analysis of codon optimization in human therapeutics.Trends Mol. Med. 2014; 20: 604-613Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar and adversely affect the performance of expression cassette elements, such as inverted terminal repeats (ITRs). Understanding the PAMP CpG threshold for human TLR9 activation, coupled with a method to quantify the TLR9 activation potential (“KTLR9”) in candidate expression cassettes, would be helpful to guide clinical vector design. Three risk factor (RF) equations were developed and used to estimate KTLR9 in 15 relevant DNA test sequences, with the results shown in Table 1. The equations progressively incorporate three attributes of DNA sequences known to activate the TLR9-MyD88 pathway. RF1 considers just the fraction (f) of total CpG dinucleotides (CpGT) divided by the nucleotide length (nt) for each DNA sequence, which ranged from 0.965% in the human genome (suppressed compared to 6.25% for random nucleotide utilization) to 9.42% in the bacterium K. peumoniae genome in the 15 sequences analyzed. RF2 multiplies RF1 by the estimated fraction of CpG dinucleotides that are unmethylated (CpGMeneg / CpGT) in each type of DNA test sequence:12Tost J. DNA methylation: an introduction to the biology and the disease-associated changes of a promising biomarker.Mol. Biotechnol. 2010; 44: 71-81Crossref PubMed Scopus (170) Google Scholar ∼0.25 for human DNA (RF2 = 0.25RF1), 1.0 for bacterial DNA (RF2 = RF1), and ∼0.95 for the viruses and rAAV vectors2Tóth 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 (AA2).Viruses. 2019; 11: 38Crossref Scopus (10) Google Scholar listed in Table 1 (RF2 = 0.95 RF1). RF3 modifies RF2 to incorporate known immune-stimulatory (S4) and -inhibitory (I4) tetranucleotide CpG motifs reported by vaccine research aiming to enhance cellular immune responses using oligonucleotide adjuvants.3Ohto U. Shibata T. Tanji H. Ishida H. Krayukhina E. Uchiyama S. Miyake K. Shimizu T. Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9.Nature. 2015; 520: 702-705Crossref PubMed Scopus (237) Google Scholar,13Bode 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,14Pohar J. Yamamoto C. Fukui R. Cajnko M.M. Miyake K. Jerala R. Benčina M. Selectivity of human TLR9 for double CpG motifs and implications for the recognition of genomic DNA.J. Immunol. 2017; 198: 2093-2104Crossref PubMed Scopus (34) Google Scholar The S4 and I4 CpG motifs were enumerated and summed for each test DNA sequence. The motif sequences selected and their TLR9 activation “weights” used for the RF3 equation in Table 2 are preliminary and directional. A broader CpG motif selection and more accurate, data-based, motif weighting factors would improve the predictive potential. A similar formula that incorporated immune-stimulatory (S6) and -inhibitory (I6) hexanucleotide CpG motifs gave a comparable range of values and the same relative ranking of the test sequences as obtained using RF3 (not shown). A normalized value for RF3 (NRF3) was calculated by dividing the RF3 value for each DNA test article by that for the human genome (0.191), i.e., the sequence assumed to represent the lowest risk of TLR9 pathway activation. The NRF3 for the complete human genome is, by definition, unity, with values ranging from 0.92 to 2.68 for selected human genes and a CpG-rich portion of chromosome 1, providing an indication of intragenomic variation. In contrast, an average NRF3 value of 20.7 was measured for three bacterial genomes known to be strongly TLR9 activating.15Dalpke A. Frank J. Peter M. Heeg K. Activation of toll-like receptor 9 by DNA from different bacterial species.Infect. Immun. 2006; 74: 940-946Crossref PubMed Scopus (131) Google Scholar Together, the human and bacterial genome data define a NRF3 range from 1 to ∼20 corresponding from negligible (−) to high (+++) values for TLR9 activation potential. The NRF3 values for the genomes of helper viruses used in rAAV production ranged from 13.2 to 28.1, demonstrating the PAMP CpG risk represented by residual helper virus DNA impurities in purified AAV preparations. While it is challenging to obtain complete expression cassette DNA sequences for clinical vectors, the availability of sequences, clinical immunotoxicity, and therapeutic outcomes for the four AAV-FIX vectors9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar listed in Table 1 provide an opportunity to further qualify the NRF3 equation. The better clinical performance of AAVSPK-FIX Padua/ss and AAV8-FIX/sc, including long-term transgene expression and lower incidences of CTLs and immunotoxicity,9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar correspond to lower NRF3 values of 3.09 and 6.80, respectively. The higher NRF3 values of 7.80 and 12.7 calculated for AAV2-FIX/ss and AAV8-FIX19/ss, respectively, correspond to vectors that gave higher immunotoxicity without durable transgene expression.9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar These data support that AAV vectors with lower NRF3 scores approaching a “humanized” value have lower immunotoxicity and better long-term clinical benefit, while those with scores above a threshold value of ∼7 are associated with deleterious immune responses not well-controlled by immune suppression, leading to loss of transgene expression. Use of such quantitative tools to evaluate TLR9 activation potential after their further refinement and validation with genome sequences from other clinical constructs represents an approach to improve AAV vectors by reducing their potential to cause immunotoxicity.Table 1TLR9 Activation Risk Factors for Selected DNA SequencesDNA Test ArticleReferenceRF1RF2RF3NRF3KTLR9HumanComplete genomeNCBI Homo sapiens GRCh380.9650.2410.1911.00−F8 geneNCBI: NG_0114030.9210.2300.1790.94−F9 geneNCBI: NC_0000230.7490.1870.1760.92−Dystrophin geneNCBI: NG_0122320.7970.1990.2001.05−Chr1 CpG-rich fragmentaChromosome 1, nucleotides 1,000,000 to 2,000,000.NCBI: NC_0000013.7040.9260.5112.68−Clinical rAAVAAVSPK-FIX Padua/ssWright9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar1.0270.9760.5903.09−AAV8-FIX/scWright9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar1.7571.6691.2986.80+AAV2-FIX/ssWright9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar2.0371.9361.4907.80++AAV8-FIX19/ssWright9Wright J.F. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar3.5303.3542.41812.7+++BacterialEscherichia coliNCBI: CP_0096857.4717.4714.68324.5+++Klebsiella pneumoniaeNCBI: FO_8349069.4219.4214.42123.1+++Staphylococcus aureusNCBI: NC_0077952.5482.5482.75014.4+++Helper VirusesAAV2NCBI: NC-0406715.8475.5554.96826.0+++Adenovirus5NCBI: AC_0000086.7176.3812.52213.2+++Autographa californicaNCBI: NC_0016236.1835.8735.36328.1+++a Chromosome 1, nucleotides 1,000,000 to 2,000,000. Open table in a new tab Table 2CpG Motifs and Equations Used to Calculate Risk FactorsMotif NameCpG Tetranucleotides IncludedKTLR9 WeightingCpGS4Σ ACGT, TCGT, CCGT+2∗CpGExCpGT - CpGS4 - CpGI4+1CpGI4Σ GCGG, CCGC, GCGC−1RF1=f[CpGT/ nt]×100%RF2=f[CpGT/ nt]×f[CpGMeneg/ CpGT]×100%RF3=f[*CpGEx+2CpGS4–CpGI4/nt]×f [CpGMeneg/ CpGT]×100% =f[CpGT+CpGS4–2CpGI4/nt]×f[CpGMeneg/CpGT]×100%NRF3=RF3(testarticle)/ RF3(humangenome) Open table in a new tab RF1=f[CpGT/ nt]×100%RF2=f[CpGT/ nt]×f[CpGMeneg/ CpGT]×100%RF3=f[*CpGEx+2CpGS4–CpGI4/nt]×f [CpGMeneg/ CpGT]×100% =f[CpGT+CpGS4–2CpGI4/nt]×f[CpGMeneg/CpGT]×100%NRF3=RF3(testarticle)/ RF3(humangenome) The author thanks Thomas Chalberg for enumeration of CpG, S4, S6, I4, and I6 motifs in the human genome (hg38 assembly) using a Python-based scripting program, Cecile Martin for science graphic artist support for Figure 1, and Maria-Grazia Roncarolo, Thomas Chalberg, and Bradley Hamilton for review of the manuscript and helpful comments.