Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates

In NHPs, high concentrations of anti-AAV antibodies significantly reduced hepatocyte transduction.

“In non-human primates (NHPs), high concentrations of anti-AAV antibodies significantly reduced the transduction of hepatocytes,29”

• cites [29] Wang L, Calcedo R, Wang H, Bell P, Grant R, Vandenberghe LH et al. The pleiotropic effects of natural AAV infections on liver-directed gene transfer in macaques. Mol Ther 2010; 18: 126–134. sourceSupported

Pre-existing antibodies correlated with decreased efficacy of gene therapy in clinical trials where administration exposes vector to serum.

“the presence of pre-existing antibodies has been correlated with decreased efficacy of gene therapy in clinical trials where the route of administration exposes vector to serum.26,30,31”

• cites [26] Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347. sourceNot checked — no full text of the source
• cites [30] Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC et al. Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 2011; 365: 2357–2365. sourcePartly supported

  The Results section (Safety and Efficacy of Peripheral-Vein Infusion of Vector; page unavailable) reports that Participant 3 had higher pre-treatment anti-AAV8 antibody levels and initially low FIX activity, raising the possibility that antibodies blocked transduction. It does not establish a correlation across clinical trials: the authors describe one participant, and his later FIX levels were consistent with successful transgene expression. (LLM, conf=0.84)

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• cites [31] Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B et al. Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase 1/2 clinical trial. J Card Fail 2009; 15: 171–181. sourcePartly supported

  The Discussion reports a potential difference in heart-failure progression among the first nine CUPID patients: both patients with pre-existing neutralizing antibodies worsened, and vector sequences were undetectable in both. But it calls the findings too preliminary to draw conclusions. This supports a possible association in one trial, not an established correlation across clinical trials. (LLM, conf=0.85)

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Transient immunosuppression is not effective at mitigating vector neutralization by pre-existing antibodies.

“While transient immunosuppression may be a promising approach for reducing cellular immune responses to gene therapy,39–41 it is not effective at mitigating vector neutralization by pre-existing antibodies.”

• cites [39] Halbert CL, Standaert TA, Wilson CB, Miller AD . Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure. J Virol 1998; 72: 9795–9805. sourceNot checked — no full text of the source
• cites [40] Jiang H, Couto LB, Patarroyo-White S, Liu T, Nagy D, Vargas JA et al. Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy. Blood 2006; 108: 3321–3328. sourcePartly supported

  The Results section on preexisting AAV8 neutralizing antibodies reports that antibodies at a titer of 1:5 neutralized the vector and prevented detectable FIX expression in rhesus macaques (page unavailable). The Discussion says the transient anti-T-cell regimen did not reliably block the antibody response to AAV capsid (page unavailable). However, the cited passages do not show a direct test of whether that regimen mitigated neutralization by antibodies already present before treatment. (LLM, conf=0.83)

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• cites [41] Manning WC, Zhou S, Bland MP, Escobedo JA, Dwarki V . Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors. Hum Gene Ther 1998; 9: 477–485. sourceNot checked — no full text of the source

AAV administration to immune privileged regions can lead to increases in neutralizing antibody titers, including after intravitreal injection in dogs and NHPs and intracranial injections in clinical trials.

“Furthermore, AAV administration to immune privileged regions can lead to increases in neutralizing antibody titers, including following intravitreal injection in dogs and non-human primates37 and following intracranial injections in several clinical trials.38”

• cites [37] Amado D, Mingozzi F, Hui D, Bennicelli JL, Wei Z, Chen Y et al. Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness. Sci Transl Med 2010; 2: 21ra16. sourcePartly supported

  The Results sections on immune responses in dogs and NHPs report increased AAV2 neutralizing-antibody titers after ocular vector administration. But the Results section on bilateral readministration specifies that the vector was injected subretinally, not intravitreally; the intravitreal injection mentioned there was ciprofloxacin. The intracranial-trial clause is attached to a different citation and is not assessed here. (LLM, conf=0.90)

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• cites [38] Mingozzi F, High KA . Immune responses to AAV in clinical trials. Curr Gene Ther 2011; 11: 321–330. sourceNot checked — no full text of the source

Subretinal delivery is limited to tissues in contact with the bleb(s).

“delivery is limited to tissues in contact with the bleb(s),18–20”

• cites [18] Aleman TS, Cideciyan A V, Sumaroka A, Windsor EAM, Herrera W, White DA et al. Retinal laminar architecture in human retinitis pigmentosa caused by Rhodopsin gene mutations. Invest Ophthalmol Vis Sci 2008; 49: 1580–1590. sourceCould not verify

  The Discussion addresses which retinal regions might be treatable in RHO-associated retinitis pigmentosa, but it does not describe a delivery area defined by a subretinal bleb. Searches for “bleb,” “subretinal,” and delivery limited to tissues in contact with a bleb found no passage supporting the claim; the available passages have no page labels. (LLM, conf=0.76)

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• cites [19] Jacobson SG, Aleman TS, Cideciyan A V, Roman AJ, Sumaroka A, Windsor EAM et al. Defining the residual vision in leber congenital amaurosis caused by RPE65 mutations. Invest Ophthalmol Vis Sci 2009; 50: 2368–2375. sourcePartly supported

  The Discussion describes a subretinal treatment zone and considers different injection sites for focal retinal therapy; the Conclusions likewise refer to selecting retinal sites for delivery. These passages support the idea that treatment is localized, but they do not state that delivery is limited specifically to tissues in contact with a bleb. Page numbers were not provided in our copy. (LLM, conf=0.78)

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• cites [20] Jacobson SG, Aleman TS, Sumaroka A, Cideciyan A V, Roman AJ, Windsor EAM et al. Disease boundaries in the retina of patients with Usher syndrome caused by MYO7A gene mutations. Invest Ophthalmol Vis Sci 2009; 50: 1886–1894. sourceNot checked — no full text of the source

Recent hemophilia clinical trials excluded patients whose neutralizing antibodies were above 1:4.

“Recent clinical trials for hemophilia have used even more stringent criteria for patient exclusion, removing patients whose neutralizing antibodies were above 1:4.31,43”

• cites [31] Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B et al. Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase 1/2 clinical trial. J Card Fail 2009; 15: 171–181. sourcePartly supported

  The Discussion (page unavailable) says the CUPID heart-failure trial screened for low or absent neutralizing antibodies and later excluded all antibody-positive patients. It does not describe a hemophilia trial or an exclusion threshold of 1:4; the titers it reports are 1:2 and <1:2. (LLM, conf=0.78)

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• cites [43] Jessup M, Greenberg B, Mancini D, Cappola T, Pauly DF, Jaski B et al. Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID): a phase 2 trial of intracoronary gene therapy of sarcoplasmic reticulum Ca2+-ATPase in patients with advanced heart failure. Circulation 2011; 124: 304–313. sourcePartly supported

  The Discussion reports that the CUPID heart-failure trial screened patients by neutralizing-antibody titer and later excluded all antibody-positive patients. It does not report an exclusion threshold above 1:4 or an exclusion policy for hemophilia trials; the qualifying titer it gives for CUPID is below 1:2. (LLM, conf=0.86)

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AAV clinical successes to date have involved delivery either to immune privileged regions or to subjects lacking apparent AAV immunological memory.

“As a result, AAV clinical successes to date have involved delivery either to immune privileged regions or to subjects lacking apparent AAV immunological memory.8–14,30,42”

• cites [8] Hauswirth WW, Aleman TS, Kaushal S, Cideciyan A V, Schwartz SB, Wang L et al. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther 2008; 19: 979–990. sourcePartly supported

  The Introduction describes this study as a subretinal AAV gene-therapy trial, and the reported results show improved visual sensitivity in the treated eyes. It therefore provides an example of ocular AAV clinical success. It does not establish that AAV clinical successes generally were confined to immune-privileged regions or to subjects lacking apparent AAV immunological memory; the Materials and Methods describe testing for anti-AAV2 responses but do not establish that broader claim. (LLM, conf=0.82)

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• cites [9] Cideciyan A V, Hauswirth WW, Aleman TS, Kaushal S, Schwartz SB, Boye SL et al. Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. Hum Gene Ther 2009; 20: 999–1004. sourceSupported
• cites [10] Bainbridge JWB, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 2008; 358: 2231–2239. sourceSupported
• cites [11] Maguire A, Simonelli F . Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 2008; 358: 2240–2248. sourceNot checked — no full text of the source
• cites [12] Maguire AM, High K a, Auricchio A, Wright JF, Pierce E a, Testa F et al. Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet 2009; 374: 1597–1605. sourceSupported
• cites [13] Bennett J, Ashtari M, Wellman J, Marshall K a, Cyckowski LL, Chung DC et al. AAV2 gene therapy readministration in three adults with congenital blindness. Sci Transl Med 2012; 4: 120ra15. sourceSupported
• cites [14] Jacobson SG, Cideciyan A V, Ratnakaram R, Heon E, Schwartz SB, Roman AJ et al. Gene therapy for leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol 2012; 130: 9–24. sourceSupported
• cites [30] Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC et al. Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 2011; 365: 2357–2365. sourceSupported
• cites [42] McPhee SWJ, Janson CG, Li C, Samulski RJ, Camp a S, Francis J et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med 2006; 8: 577–588. sourceNot checked — no full text of the source

Pre-existing antibodies inhibit AAV transduction broadly documented following mouse, rabbit, NHP, and human gene therapy administrations.

“The observation that pre-existing antibodies inhibit AAV transduction has been broadly documented following mouse, rabbit, non-human primate, and human gene therapy administrations.31,57–59”

• cites [31] Jaski BE, Jessup ML, Mancini DM, Cappola TP, Pauly DF, Greenberg B et al. Calcium upregulation by percutaneous administration of gene therapy in cardiac disease (CUPID Trial), a first-in-human phase 1/2 clinical trial. J Card Fail 2009; 15: 171–181. sourcePartly supported

  The Discussion (page not provided) says preexisting neutralizing antibodies can block viral-vector entry, and the paper reports suggestive findings in two antibody-positive CUPID patients. Its References include a mouse transduction study, but our copy does not document the claimed rabbit or non-human-primate findings; the Discussion also says its human data are too preliminary for conclusions. (LLM, conf=0.84)

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• cites [57] Lin J, Calcedo R, Vandenberghe LH, Figueredo JM, Wilson JM . Impact of preexisting vector immunity on the efficacy of adeno-associated virus-based HIV-1 Gag vaccines. Hum Gene Ther 2008; 19: 663–669. sourcePartly supported

  The Introduction (page unavailable) states that even low circulating antibody titers diminish AAV2 transduction in gene-therapy settings. The Results and Discussion (pages unavailable) show that human or macaque antibodies transferred into mice impair AAV vaccine responses. The paper does not document rabbit administrations or demonstrate inhibited transduction following administrations in each of the four named species; its own experiments measured vaccine responses in mice. (LLM, conf=0.85)

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• cites [58] Halbert CL, Standaert TA, Aitken ML, Alexander IE, Russell DW, Miller AD . Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration. J Virol 1997; 71: 5932–5941. sourcePartly supported

  The indexed passage on rabbit airway transduction (section and page unavailable) reports that AAV readministration produced no additional transduction and that this correlated with the appearance of neutralizing antibodies. It does not establish that pre-existing antibodies caused the inhibition. This paper addresses rabbits; the other species in the multiply cited sentence are not supported by this source. (LLM, conf=0.86)

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• cites [59] Wang L, Calcedo R, Bell P, Lin J, Grant RL, Siegel DL et al. Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. Hum Gene Ther 2011; 22: 1389–1401. sourcePartly supported

  The Results section reports that pre-existing neutralizing antibodies above a 1:10 titer substantially inhibited AAV8 transfer to macaque liver, and the Introduction describes earlier AAV transduction inhibition. But the Introduction’s mouse-and-rabbit example concerns adenovirus, not AAV; this paper therefore does not support the claim of documented AAV inhibition in rabbits. Page numbers were not available in our copy. (LLM, conf=0.85)

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The higher initial AAV8/AAV9 titers are most likely due to high prevalence of natural AAV8 and AAV9 infections in rhesus macaques.

“most likely due to the high prevalence of natural AAV8 and AAV9 infections in rhesus macaques.53,54”

• cites [53] Gao G-P, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM . Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002; 99: 11854–11859. sourcePartly supported

  The Results (page unavailable) report high-titer antibodies to AAV8 in 45% of rhesus monkeys across the colonies studied, supporting evidence of prevalent AAV8 exposure. The paper studies AAV7 and AAV8, not AAV9, and does not link natural infections to the higher initial AAV8/AAV9 titers in the claim. (LLM, conf=0.88)

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• cites [54] Nieto K, Stahl-Hennig C, Leuchs B, Müller M, Gissmann L, Kleinschmidt JA . Intranasal vaccination with AAV5 and 9 vectors against human papillomavirus type 16 in rhesus macaques. Hum Gene Ther 2012; 23: 733–741. sourceNot checked — no full text of the source

AAV8 and AAV9 capsids have the highest sequence similarity of the four capsids studied (85.7% amino acid sequence identity).

“AAV8 and AAV9 capsids have the highest sequence similarity of any of the four capsids studied (85.7% amino acid sequence identity)55”

• cites [55] Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X et al. Clades of adeno-associated viruses are widely disseminated in human tissues. J Virol 2004; 78: 6381–6388. sourcePartly supported

  Table 3 (Results and Discussion; page not provided) reports 14.3% cap amino acid differences between AAV8 and AAV9, corresponding to 85.7% identity. The paper compares more than four capsids and does not establish that AAV8 and AAV9 are the most similar of the citing study’s four; its table includes pairs with fewer differences. (LLM, conf=0.85)

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In macaque livers following intravenous AAV8, neutralizing antibody titers higher than 1:10 substantially decreased GFP expression in hepatocytes.

“In a study monitoring GFP expression in macaque livers following intravenous AAV8 vector administration, neutralizing antibody titers higher than 1:10 substantially decreased GFP expression in hepatocytes.59”

• cites [59] Wang L, Calcedo R, Bell P, Lin J, Grant RL, Siegel DL et al. Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. Hum Gene Ther 2011; 22: 1389–1401. sourceSupported

AAV5 has the most distinct sequence of the capsids (61.2% identity to AAV2; AAV8 83.6%; AAV9 82.7%).

“AAV5 has the most distinct sequence of the capsids (AAV5: 61.2% amino acid sequence identity to AAV2, AAV8: 83.6% amino acid sequence identity to AAV2, AAV9: 82.7% sequence identity to AAV2),55,56”

• cites [55] Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X et al. Clades of adeno-associated viruses are widely disseminated in human tissues. J Virol 2004; 78: 6381–6388. sourceSupported
• cites [56] Bantel-Schaal U, Delius H, Schmidt R, zur Hausen H . Human adeno-associated virus type 5 is only distantly related to other known primate helper-dependent parvoviruses. J Virol 1999; 73: 939–947. sourcePartly supported

  The capsid-alignment results and Table 1 support that AAV5 is distinct from the serotypes this paper compared. But Table 1 gives 58.3% identity for AAV5 versus AAV2, not 61.2%, and the paper does not report the claimed AAV8 or AAV9 comparisons (page numbers unavailable in our copy). (LLM, conf=0.90)

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Humoral immunological memory can inhibit or block AAV gene delivery for routes of administration that expose vector to host antibodies.

“However, humoral immunological memory can inhibit or block AAV gene delivery for routes of administrations that expose vector to host antibodies.57–59”

• cites [57] Lin J, Calcedo R, Vandenberghe LH, Figueredo JM, Wilson JM . Impact of preexisting vector immunity on the efficacy of adeno-associated virus-based HIV-1 Gag vaccines. Hum Gene Ther 2008; 19: 663–669. sourceSupported
• cites [58] Halbert CL, Standaert TA, Aitken ML, Alexander IE, Russell DW, Miller AD . Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration. J Virol 1997; 71: 5932–5941. sourcePartly supported

  The Abstract (page not provided) reports that AAV readministration in rabbit airways produced no additional transduction, correlated with the appearance of neutralizing antibodies. This supports antibody-associated inhibition of repeat delivery, but the paper does not establish the broader claim for all administration routes that expose vectors to host antibodies or demonstrate that immunological memory caused the failure. (LLM, conf=0.84)

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• cites [59] Wang L, Calcedo R, Bell P, Lin J, Grant RL, Siegel DL et al. Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. Hum Gene Ther 2011; 22: 1389–1401. sourceSupported

Mapping linear and conformational epitopes for neutralizing antibody binding and structural analysis of docking sites for a murine IgG2a antibody identified immunogenic AAV capsid regions that could be mutated to create variants with reduced neutralization by mouse and human antibodies in vitro.

“Mapping of linear and conformational epitopes responsible for neutralizing antibody binding and in silico structural analysis of potential docking sites for a murine IgG2a antibody have led to the discovery of immunogenic regions of the AAV capsid that could been mutated to develop variants with reduced neutralization by mouse and human antibodies in vitro.73–75”

• cites [73] Lochrie MA, Tatsuno GP, Christie B, McDonnell JW, Zhou S, Surosky R et al. Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization. J Virol 2006; 80: 821–834. sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

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• cites [74] Moskalenko M, Chen L, van Roey M, Donahue BA, Snyder RO, McArthur JG et al. Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure. J Virol 2000; 74: 1761–1766. sourceNot checked — no full text of the source
• cites [75] Wobus CE, Hügle-Dörr B, Girod A, Petersen G, Hallek M, Kleinschmidt JA . Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. J Virol 2000; 74: 9281–9293. sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

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Post-injection antibodies cross-reacted with other AAV serotypes.

“the antibodies were also cross-reactive against other AAV serotypes.”

• Also stated in: § Cross-Reactivity of Antibodies to Various AAV Serotypes Pre-Infection vs. Post-Infection · plain

  “anti-AAV5, anti-AAV8, and anti-AAV9 antibody titers also increase following administration of any of the other serotypes (Figure 5a-h).”

Intravitreal administration increased anti-AAV antibodies regardless of capsid serotype, transgene, or virus dosage.

“Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene, or dosage of virus injected.”

• Also stated in: § Discussion · plain

  “Anti-AAV antibody production following intravitreal injection increased regardless of the capsid serotype, transgene, amount of virus, or number of eyes injected.”

Neutralizing antibody levels in serum correlated with those in vitreal fluid.

“Furthermore, neutralizing antibody levels in serum correlated with those in vitreal fluid”

• Also stated in: § Comparison of AAV2 Neutralization by Serum vs. Vitreous Fluid · hedged

  “in over half of the samples tested, vitreous fluid and serum samples had the same level of AAV2 neutralization, and in others the levels were within an order of magnitude (Figure 3), indicating that the level of anti-AAV antibodies in the serum is positively correlated with anti-AAV antibody levels in the vitreous fluid (R=0.30).”

For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the amount of virus delivered most significantly affected anti-AAV2 antibody production.

“For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered.”

• Also stated in: § Increase of Anti-AAV2 Antibodies Pre-Infection vs. Post-Infection · plain

  “for the seven monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies is the amount of virus delivered (Figure 1h – total virus > 2×1012 vg: 200-500 fold increase vs. Figure 1i – total virus < 2×1012 vg: 10-50 fold increase; p=0.017).”

Pre-existing neutralizing antibody titers in serum correlated strongly (R=0.76) with weak, decaying, or no transgene expression following intravitreal AAV.

“Moreover, the presence of pre-existing neutralizing antibody titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying, or no transgene expression following intravitreal administration of AAV.”

• Also stated in: § Effect of Pre-Injection Anti-AAV2 Antibodies on Expression · hedged

  “At any rate, these data suggest that systemic anti-AAV antibodies correlate (R=0.7639) with a loss of vector transduction in other body fluid compartments such as the vitreous.”

Post-injection antibody titers were highest against the administered serotype.

“In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were”

• Also stated in: § Discussion · plain

  “As anticipated, the downstream antibody response is stronger against the administered serotype than other AAV serotypes”

Monkey 108 (AAV5 and AAV9) had a larger anti-AAV2 response than Monkey 012 (AAV5 only).

“Monkey 108 (injected with both AAV5 and AAV9 – Figure 1c, black circles) had a larger anti-AAV2 response than Monkey 012 (only injected with AAV5 – Figure 1c, gray triangles)”

For all nine animals, anti-AAV2 antibody titers of vitreous fluid from right and left eyes were equal.

“For all nine animals, the anti-AAV2 antibody titers of the vitreous fluid from the right and left eyes were equal (Figure 3).”

Even when one eye received much larger amount of virus, neutralizing titers in both eyes at later time were equal.

“even when one eye received a much larger amount of virus (as is the case with Monkey 108 and Monkey 622), the neutralizing titers in both eyes at the later time were equal.”

Anti-AAV2 antibody titers increased in all animals following injection regardless of capsid serotype, transgene, dosage, or unilateral vs bilateral injection.

“Anti-AAV2 antibody titers increased in all animals following injection regardless of the capsid serotype, transgene, dosage of virus, or unilateral vs. bilateral injection.”

Single-eye and double-eye injections showed similar magnitudes of anti-AAV2 NAB response (10-500 vs 50-250 fold; p=0.996).

“Sera samples from Monkeys 002, 109, 110, 906, and 909 (injected in one eye – Figure 1f) and Monkeys 014, 736, and 902 (injected in both eyes– Figure 1g) showed similar magnitudes of anti-AAV2 NAB response (Figure 1f – single eye: 10-500 fold increase post-injection vs. Figure 1g – double eye: 50-250 fold increase post-injection; p=0.996).”

Small sequence changes to a serotype (wild-type AAV2 vs 7m8) do not significantly affect the antibody response in the seven animals in which these vectors were administered.

“small sequence changes to a serotype (i.e. wild-type AAV2 vs. 7m8) do not significantly affect the antibody response in the seven animals in which these vectors were administered”

NAB reactivities against AAV5, AAV8, or AAV9 were not significantly different following administration of wild-type AAV2 capsids, AAV2 tyrosine 4YF mutant capsids, or 7m8 capsids.

“the NAB reactivities against AAV5, AAV8, or AAV9 were not significantly different following administration of wild-type AAV2 capsids, AAV2 tyrosine 4YF mutant capsids, or 7m8 capsids (Figure 5a-d).”

There was no significant difference in serum dilution at which AAV2 tyrosine 4YF mutant was ~50% neutralized compared to 7m8 and wild-type AAV2.

“there was no significant difference in the serum dilution at which it was ∼50% neutralized compared to the 7m8 capsid and wild-type AAV2.”

Long-term data from several animals and three-year monitoring of Monkey 708 demonstrated that antibodies can persist for very long periods after gene therapy administration.

“long-term data from several animals (Monkeys 002, 012, 013, 014, and 902) – coupled with three year monitoring of Monkey 708 (Figure 4) – demonstrated that antibodies can persist for very long periods after gene therapy administration.”

Monkeys injected with AAV2 generated post-administration antibodies that neutralized AAV2 slightly more potently than monkeys injected with other serotypes (10-50 fold vs 2.5-50 fold increase).

“monkeys injected with AAV2 generated post-administration antibodies that neutralized AAV2 slightly more potently than monkeys injected with other serotypes (Figure 1b – wild-type AAV2: 10-50 fold increase post-injection vs. Figure 1c – wild-type AAV9 and/or AAV5: 2.5-50 fold increase post-injection).”

For monkeys undergoing intravitreal administrations involving AAV2, 7m8, or AAV2 tyrosine 4YF mutant capsids, serum neutralizing antibody titers of 1:10 or greater resulted in weak or no transgene expression.

“We found that for monkeys undergoing intravitreal administrations of vector involving AAV2, 7m8, or AAV2 tyrosine 4YF mutant capsids, the presence of neutralizing antibody titers of 1:10 or greater in the serum resulted in weak or no expression of the transgene (Table 1).”

In 9 of 11 monkeys tested, initial antibody titers against AAV8 and AAV9 were higher than antibody titers against AAV2.

“In 9 of 11 monkeys tested, initial antibody titers against AAV8 and AAV9 were higher than antibody titers against AAV2”

AAV8 and AAV9 showed consistent magnitudes of anti-AAV8 and anti-AAV9 antibody induction in most monkeys.

“and showed consistent magnitudes of anti-AAV8 and anti-AAV9 antibody induction in most monkeys.”

Intravitreal delivery of AAV vectors to monkeys raised the neutralizing antibody titers in their serum.

“In addition, increases in neutralizing antibody titer were seen in the sera of these monkeys following intravitreal administration of AAV vectors.”

AAV5 had the least predictable antibody response in monkeys administered other serotypes.

“and consequently had the least predictable antibody response in monkeys administered other serotypes.”

In monkeys that received wild-type AAV2 and/or an AAV2 mutant, the total amount of virus administered was the factor most strongly associated with anti-AAV2 antibody production.

“The variable that most significantly affected the production of anti-AAV2 antibodies in monkeys injected with wild-type AAV2 and/or an AAV2 mutant was the total amount of virus delivered.”

7m8 was neutralized at the same serum dilution as wild-type AAV2 capsid using either pooled human IVIG or individual monkey sera.

“For example, 7m8 was neutralized at the same serum dilution as wild-type AAV2 capsid using either pooled human IVIG or individual monkey sera.”

High doses of AAV2 viral capsids induce greater anti-AAV5, anti-AAV8, and anti-AAV9 antibody responses.

“high doses of AAV2 viral capsids also induce greater anti-AAV5, anti-AAV8, and anti-AAV9 antibody responses in these animals (Figure 6b, c).”

Monkey 110, injected with 7m8 then AAV9, did not follow the trend of progressive increase.

“Interestingly, Monkey 110, which was injected with a 7m8 capsid, then an AAV9 capsid, did not follow this trend (Figure 6c).”

Neutralizing titers in the range of 1:25 to 1:100 occasionally corresponded to weak transgene expression or transgene expression that decayed after initial observation.

“Additionally, neutralizing titers in the range of 1:25 to 1:100 occasionally corresponded to weak transgene expression or transgene expression that decayed after initial observation.”

AAV5 injected 101 days after AAV2 led 29 days later to a 2.5-fold increase in anti-AAV2 antibody titer.

“AAV5 was injected 101 days after AAV2, which led 29 days later to a 2.5-fold increase in anti-AAV2 antibody titer (Figure 6a)”

Monkey 002 had a 10-fold increase in anti-AAV2 antibody titer 46 days following AAV2 injection.

“Monkey 002 (Figure 6a) had a 10-fold increase in anti-AAV2 antibody titer 46 days following injection of AAV2.”

Antibody titers further increased following a second AAV administration.

“Antibody titers also further increased following a second AAV administration.”

Analysis of sera from non-human primates before and after intravitreal injections of various AAV capsids showed clear increases in anti-vector antibodies after intravitreal administration.

“analysis of sera collected from non-human primates before and after intravitreal injections of various AAV capsids showed clear increases in anti-vector antibodies following intravitreal administration”

Serum neutralizing antibody titers generally correlated well with vitreous fluid titers.

“in general serum NAB titers correlated well with vitreous fluid titers.”

The presence of anti-AAV antibodies is correlated with weak, degenerating, or absent transgene expression in the eye.

“Additionally, the presence of these anti-AAV antibodies correlates to weak, degenerating, or no transgene expression in the eye.”

Serum measurements are predictive of vitreous fluid NAB titers.

“and that serum measurements are predictive of vitreous fluid NAB titers.”

Intravitreal administration exposes AAV capsid epitopes to the adaptive immune system.

“demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system”

• Also stated in: § Discussion · hedged

  “Furthermore, intravitreal administration resulted in an increase in anti-AAV antibodies in serum, indicating that this route presents AAV capsid antigens to the adaptive immune system.”

Extremely high serum neutralizing antibody titers are accompanied by reduced AAV vector transduction in rat brain after intracranial injection or mouse eye after intravitreal injection.

“the presence of extremely high titers of neutralizing antibodies in serum are accompanied by reduced AAV vector transduction in the rat brain following intracranial injection or in the mouse eye following intravitreal injection.35,36”

• cites [35] Sanftner LM, Suzuki BM, Doroudchi MM, Feng L, McClelland A, Forsayeth JR et al. Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV. Mol Ther 2004; 9: 403–409. sourceNot checked — no full text of the source
• cites [36] Li Q, Miller R, Han P-Y, Pang J, Dinculescu A, Chiodo V et al. Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential. Mol Vis 2008; 14: 1760–1769.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

It is unlikely that the 7m8 modification results in a different exposure of antibody binding epitopes.

“Since AAV2 and 7m8 are thus equally neutralized by NHP and human antibodies, it to be unlikely that the 7m8 modification results in a different exposure of antibody binding epitopes.”

The LCA clinical trial excluded patients with neutralizing antibodies to AAV2 above 1:1000.

“During the LCA clinical trial, patients were excluded “if immunological studies show presence of neutralizing antibodies to AAV2 above 1:1000.”11”

• cites [11] Maguire A, Simonelli F . Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 2008; 358: 2240–2248. sourceNot checked — no full text of the source

The finding that anti-AAV2 titers increase after intravitreal injection is consistent with previous rodent reports indicating the vitreous is not as immune-privileged as subretinal delivery for AAV or adenovirus.

“This finding is consistent with previous reports in rodents indicating that the vitreous is not as immune-privileged a route of administration as subretinal delivery for either AAV or adenovirus delivery.47,48”

• cites [47] Martin KRG, Klein RL, Quigley HA . Gene delivery to the eye using adeno-associated viral vectors. Methods 2002; 28: 267–275. sourceNot checked — no full text of the source
• cites [48] Hoffman LM, Maguire AM, Bennett J . Cell-mediated immune response and stability of intraocular transgene expression after adenovirus-mediated delivery. Invest Ophthalmol Vis Sci 1997; 38: 2224–2233.Not checked — no full text of the source

Minimal differences in neutralizing antibody titer between wtAAV2 and 7m8 are expected given that their capsid proteins differ by <10 amino acids.

“These minimal differences in neutralizing antibody titer between wtAAV2 and 7m8 are to be expected, given that their capsid proteins differ by <10 amino acids.”

Pre-existing immunity can hinder transduction by AAV vectors in immune-privileged sites such as the eye and brain.

“Weak and decaying transgene expression following intravitreal administration of AAV vectors to monkeys harboring anti-AAV antibodies provides more evidence that pre-existing immunity can challenge transduction of AAV vectors in immune privileged regions, such as the eye and brain.35”

• cites [35] Sanftner LM, Suzuki BM, Doroudchi MM, Feng L, McClelland A, Forsayeth JR et al. Striatal delivery of rAAV-hAADC to rats with preexisting immunity to AAV. Mol Ther 2004; 9: 403–409. sourceNot checked — no full text of the source

• AssumptionRests on an assumption: The eye is an immune-privileged site.

Due to high sequence similarity between AAV2, AAV8, and AAV9, administration of one of these vectors led to increases in antibody titers against all.

“however, due to the high sequence similarity between AAV2, AAV8, and AAV9, administration of one of these vectors led to increases in antibody titers against all.”

A capsid decoy strategy reported by Mingozzi et al. showed that mixing gene therapy vectors with empty AAV2-based capsid particles carrying mutations that abolish primary cell receptor binding enhanced transduction of the gene therapy vector when low to moderate levels of neutralizing antibodies were present.

“Moreover, a capsid decoy strategy by Mingozzi et al. demonstrated that mixing gene therapy vectors with empty AAV2-based capsid particles with mutations that ablate primary cell receptor binding could enhance transduction of the gene therapy vector in the presence of low to moderate levels of neutralizing antibodies.76”

• cites [76] Mingozzi F, Anguela XM, Pavani G, Chen Y, Davidson RJ, Hui DJ et al. Overcoming preexisting humoral immunity to AAV using capsid decoys. Sci Transl Med 2013; 5: 194ra92. sourceNot checked — no full text of the source

These antibody-evasion properties also produced enhanced in vivo transduction: the variants transduced liver, heart, and muscle significantly better than AAV2 in mice passively immunized with human antibodies.

“These antibody neutralization properties also led to enhanced transduction in vivo, where AAV variants were capable of significantly higher transduction in the liver, heart and muscle than AAV2 in mice passively immunized with human antibodies.”

• cites [68] Maheshri N, Koerber JT, Kaspar BK, Schaffer DV . Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat Biotechnol 2006; 24: 198–204. sourceNot checked — no full text of the source
• cites [69] Koerber JT, Jang J-H, Schaffer DV . DNA shuffling of adeno-associated virus yields functionally diverse viral progeny. Mol Ther 2008; 16: 1703–1709. sourceNot checked — no full text of the source
• cites [70] Bartel MA, Hwang B-Y, Stone D, Koerber JT, Couto L, Mingozzi F et al. Directed evolution of aav for enhanced evasion of human neutralizing antibodies. American Society of Gene and Cell Therapy’s 15th Annual Meeting 2012: Philadelphia, PA, USA S140.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [71] Perabo L, Endell J, King S, Lux K, Goldnau D, Hallek M et al. Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus. J Gene Med 2006; 8: 155–162. sourceNot checked — no full text of the source
• cites [72] Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol 2008; 82: 5887–5911. sourceNot checked — no full text of the source

AAV variants generated by directed evolution tolerated up to 35-fold higher concentrations of pooled human antibodies in vitro than AAV2 did.

“New AAV variants created through directed evolution withstood up to 35-fold higher in vitro concentrations of pooled human antibodies than AAV2.”

• cites [68] Maheshri N, Koerber JT, Kaspar BK, Schaffer DV . Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat Biotechnol 2006; 24: 198–204. sourceNot checked — no full text of the source
• cites [69] Koerber JT, Jang J-H, Schaffer DV . DNA shuffling of adeno-associated virus yields functionally diverse viral progeny. Mol Ther 2008; 16: 1703–1709. sourceNot checked — no full text of the source
• cites [70] Bartel MA, Hwang B-Y, Stone D, Koerber JT, Couto L, Mingozzi F et al. Directed evolution of aav for enhanced evasion of human neutralizing antibodies. American Society of Gene and Cell Therapy’s 15th Annual Meeting 2012: Philadelphia, PA, USA S140.Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [71] Perabo L, Endell J, King S, Lux K, Goldnau D, Hallek M et al. Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus. J Gene Med 2006; 8: 155–162. sourceNot checked — no full text of the source
• cites [72] Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA et al. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol 2008; 82: 5887–5911. sourceNot checked — no full text of the source