The AAV2.7m8 capsid packages a higher degree of heterogeneous vector genomes than AAV2

The quote only partly states this claim.

The AAV2.7m8 capsid packages a higher proportion of heterogeneous or non-unit-length vector genomes than the AAV2 capsid across multiple vector genome designs.

“In this report, we found that AAV2.7m8 packages vector genomes with a higher degree of heterogeneity than AAV2.”

• Also stated in: § INTRODUCTION · plain

  “Using AAV Genome Population sequencing (AAV-GPseq) [29], we found that AAV2.7m8 packaged a higher degree of non-unit length genomes than AAV2.”

• Also stated in: § AAV2.7m8 preparations consistently yield undefined bands and fewer full-length genomes · boosted

  “These results demonstrate that vectors packaged with the AAV2.7m8capsid tended to package a higher degree of non-unit length forms.”

• cites [29] Tai PWL, Xie J, Fong K, Seetin M, Heiner C, Su Q, et al. Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras. Mol Ther Methods Clin Dev. 2018;9:130–41. sourceCould not verify

  The Results section on heterogeneous vector genomes reports different proportions of full-length genomes for three vector designs, but it does not compare AAV2.7m8 with AAV2 capsids. Searches for AAV2.7m8 and a capsid-based comparison found no passage addressing the claimed difference. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

May combine two claims.

IBA1 staining marks microglia and is used as a marker for immune responses in AAV-treated eyes.

“To evaluate immune responses toward vector treatment, we stained-cryosectioned retinas with anti-IBA1 (Fig. 4), which stains microglia, the resident immune cells of the retina that is used as a marker for immune responses in AAV-treated eyes [9,13,45].”

• cites [9] Xiong W, Wu DM, Xue Y, Wang SK, Chung MJ, Ji X, et al. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc Natl Acad Sci USA. 2019;116:5785–94. sourceSupported
• cites [13] Cheng SY, Luo Y, Malachi A, Ko J, Su Q, Xie J, et al. Low-dose recombinant adeno-associated virus-mediated inhibition of vascular endothelial growth factor can treat neovascular pathologies without inducing retinal vasculitis. Hum Gene Ther. 2021;32:649–66. sourceSupported
• cites [45] Chan YK, Wang SK, Chu CJ, Copland DA, Letizia AJ, Costa Verdera H, et al. Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Sci Transl Med. 2021;13:eabd3438. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

AAV2.7m8 vectors produce lower packaging titers than other serotypes, consistent with prior literature.

“Although the 7m8 modification on AAV2 shows significant improvement in retinal transduction, we have found that the capsid inherently produces lower titers compared to other serotypes, which is consistent with the literature [21].”

• cites [21] Westhaus A, Cabanes-Creus M, Rybicki A, Baltazar G, Navarro RG, Zhu E, et al. High-throughput in vitro, ex vivo, and in vivo screen of adeno-associated virus vectors based on physical and functional transduction. Hum Gene Ther. 2020;31:575–89. sourceCould not verify

  In Results and Discussion, under “Preparation of the AAV Testing Kit mix” (page unavailable), the paper includes AAV7m8 among the tested variants and reports that their titers differed, but it does not give AAV7m8’s individual titer in the available text. I searched for AAV7m8 production yield and pre-dilution titer; neither the supplied passages nor the additional passages establish that it produced lower titers than other serotypes. The paper identifies AAVAnc80, not AAV7m8, as the lowest-titer variant in this test. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Cytokine factors increase in the mouse retina after intravitreal delivery of AAV5 and AAV8 capsids.

“For instance, cytokine factors increase in the mouse retina after IVT delivery of AAV5 and AAV8 capsids [9].”

• cites [9] Xiong W, Wu DM, Xue Y, Wang SK, Chung MJ, Ji X, et al. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc Natl Acad Sci USA. 2019;116:5785–94. sourcePartly supported

  The Results section, “Microglia Activation Is Associated with AAV-Induced Toxicity” (page unavailable), reports increased retinal TNF-α and IL-1β RNA after infection with toxic AAV vectors, but not significant increases in IL-6 or IFN-γ. The paper also tests AAV5 and AAV8, but its “AAV Injection” methods specify subretinal injection, not intravitreal delivery; it does not establish cytokine increases after intravitreal delivery of both capsid types. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

The oversized species likely represent unresolved self-complementary genomes that survived Benzonase and DNase treatment.

“These species likely represent unresolved self-complementary genomes that may have survived the Benzonase endonuclease and DNase treatment steps [44].”

• cites [44] Hirata RK, Russell DW. Design and packaging of adeno-associated virus gene targeting vectors. J Virol. 2000;74:4612–20. sourcePartly supported

  The Results (page not available) identify packaged dimer-sized genomes with an inverted, self-complementary structure and report that they remained detectable after extensive micrococcal nuclease digestion. The paper does not identify the citing document’s oversized species as unresolved genomes or address their survival of Benzonase and DNase treatment. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Several reports have shown immune responses and/or toxicities from genomic contaminants that may result in the expression of unintended epitopes.

“Several reports have shown immune responses and/or toxicities from genomic contaminants that may result in the expression of unintended epitopes [49][50][51].”

• cites [49] Li C, Goudy K, Hirsch M, Asokan A, Fan Y, Alexander J, et al. Cellular immune response to cryptic epitopes during therapeutic gene transfer. Proc Natl Acad Sci USA. 2009;106:10770–4. sourceNot checked — no full text of the source
• cites [50] Keiser MS, Ranum PT, Yrigollen CM, Carrell EM, Smith GR, Muehlmatt AL, et al. Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain. Nat Med. 2021;27:1982–9. sourceNot checked — no full text of the source
• cites [51] Brimble MA, Cheng PH, Winston SM, Reeves IL, Souquette A, Spence Y, et al. Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps. Mol Ther Methods Clin Dev. 2022;24:280–91. sourceSupported

May combine two claims.

Intraocular rAAV administration has been shown to trigger vasculitis or uveitis, limiting therapeutic efficacy and risking patient complications.

“Namely, rAAV administration via intraocular injection has been shown to trigger vasculitis or uveitis [9], limiting therapeutic efficacies for vectors and risking complications for patients [10].”

• cites [9] Xiong W, Wu DM, Xue Y, Wang SK, Chung MJ, Ji X, et al. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc Natl Acad Sci USA. 2019;116:5785–94. sourcePartly supported

  The Abstract (page unavailable) reports intraocular inflammation after subretinal AAV injection, including in five of eight higher-dose trial participants. It does not identify those events as vasculitis or uveitis; the Discussion mentions uveitis only in connection with clinical grading scales. The separate claim about therapeutic efficacy and patient complications carries citation [10], not this source's citation [9]. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [10] Chandler LC, McClements ME, Yusuf IH, Martinez-Fernandez de la Camara C, MacLaren RE, Xue K. Characterizing the cellular immune response to subretinal AAV gene therapy in the murine retina. Mol Ther Methods Clin Dev. 2021;22:52–65. sourceSupported

Classified as probably not a substantial claim. May combine two claims.

In the normal eye, microglia are radially distributed from the ganglion cell layer interface and are mainly colonized at the inner plexiform layer and outer plexiform layer.

“In the normal eye, microglia are radially distributed from the GCL interface and are mainly colonized at the inner plexiform layer (IPL) and OPL [46].”

• cites [46] Murenu E, Gerhardt MJ, Biel M, Michalakis S. More than meets the eye: the role of microglia in healthy and diseased retina. Front Immunol. 2022;13:1006897. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Multiple variables can contribute to retinal toxicity following gene therapy, including endotoxin in vector stocks, high empty/full capsids, the promoter driving transgene expression, the route of administration, and the vector dose.

“Multiple variables can contribute to retinal toxicity following gene therapy: the presence of endotoxin in vector stocks, high empty/full of capsids, the promoter that is used to drive transgene expression, the route of administration, and the vector dose [9,15,47].”

• cites [9] Xiong W, Wu DM, Xue Y, Wang SK, Chung MJ, Ji X, et al. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc Natl Acad Sci USA. 2019;116:5785–94. sourcePartly supported

  The Discussion (page not provided) identifies promoter and viral dose as the strongest correlates of toxicity and suggests that high endotoxin levels or empty capsids may be problematic in other cases. It mentions differing injection routes as a reason studies are difficult to compare, but does not establish that route contributes to retinal toxicity. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [15] Timmers AM, Newmark JA, Turunen HT, Farivar T, Liu J, Song C, et al. Ocular inflammatory response to intravitreal injection of adeno-associated virus vector: relative contribution of genome and capsid. Hum Gene Ther. 2020;31:80–9. sourceNot checked — no full text of the source
• cites [47] Tummala G, Crain A, Rowlan J, Pepple KL. Characterization of gene therapy associated uveitis following intravitreal adeno-associated virus injection in mice. Invest Ophthalmol Vis Sci. 2021;62:41. sourcePartly supported

  The Results (page not provided) identify lipopolysaccharide contamination, empty capsids, and high dose as possible contributors to ocular inflammation, while the Discussion reports retinal toxicity associated with certain promoters after subretinal injection. The paper also discusses administration route, but it does not establish that all the listed variables contribute to retinal toxicity; its own experiments primarily assess uveitis after intravitreal injection. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Not all peptide inserts, including 7-mer sequences, at the 587-588 position are amenable to capsid assembly.

“The fact that that not all peptide inserts (including 7-mer sequences) at the 587-588 position are amenable to capsid assembly, is well documented [59,60].”

• cites [59] Zhu D, Brookes DH, Busia A, Carneiro A, Fannjiang C, Popova G, et al. Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy. Sci Adv. 2024;10:eadj3786. sourcePartly supported

  The Results section (“AAV5–7-mer peptide insertion library preparation and packaging selection”; page not provided) reports that more than 50% of variants in its 7-mer insertion library fail to package into viable capsids. However, that library inserts peptides at position 575–577, not the claimed 587–588 position, so the source does not establish the site-specific claim. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [60] Douar AM, Poulard K, Danos O. Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid. Virology. 2003;309:203–8. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing).

Capsids with lower stabilities correlate with improved transduction, owing to more efficient uncoating and release of vector genomes.

“Alternatively, it has been observed that capsids with lower stabilities correlate with improved transduction, owning to more efficient uncoating and release of vector genomes [62,63].”

• cites [62] Pavlou M, Schon C, Occelli LM, Rossi A, Meumann N, Boyd RF, et al. Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders. EMBO Mol Med. 2021;13:e13392. sourcePartly supported

  The Results (Characterisation of AAV2.GL and AAV2.NN; page not provided) report that the better-performing variants disassembled at a lower temperature than AAV2, suggesting genome release under less harsh conditions. The assay measures uncoating indirectly; the paper does not establish that more efficient uncoating caused their improved transduction or demonstrate a general correlation across capsids. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [63] Rode L, Bar C, Gross S, Rossi A, Meumann N, Viereck J, et al. AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes. Mol Ther. 2022;30:3601–18. sourceSupported

May combine two claims.

For ssCB6-FLuc, AAV2-packaged vectors predominantly had full-length 3.2 kb reads, while AAV2.7m8-packaged vectors predominantly had short reads under 2 kb.

“The predominant read species from the AAV2-packaged vectors was associated with the full-length 3.2-kb genome; while short, <2-kb reads were the dominant species for the AAV2.7m8-packagedvectors.”

May combine two claims.

Transgene-related inflammation in deep retinal layers depends on the presence of the transgene and is not solely due to a capsid-related immune response.

“Furthermore, we demonstrated that transgene-related inflammation in deep retinal layers is dependent on the presence of the transgene and not solely due to a capsid-related immune response.”

May combine two claims.

For ssCB6-Egfp packaged with AAV2.7m8, short reads under 2 kb were highly abundant and the expected 2.3 kb reads were in the minority.

“Interestingly, reads from the vector packaged with AAV2.7m8 exhibited a high abundance of short <2 kb reads (Fig. 3d).”

• Also stated in: § AAV-GPseq reveals that AAV2.7m8-packaged vector genomes have a higher degree of truncated genomes than AAV2-packaged vectors · plain

  “Furthermore, the expected 2.3-kb length reads were in the minority (Fig. 3d).”

Probably not a substantial claim: may not pass S3 (load-bearing).

AAV-GPseq detected a high abundance of oversized reads of about 6.4 kb, especially for the AAV2 vector.

“Curiously, we detected a high abundance of reads representing oversized genomes (~6.4 kb) (Fig. 3f), especially for the AAV2 vector.”

May combine two claims.

For ssCB6-Egfp packaged with AAV2, reads predominantly spanned ITR-to-ITR and showed two populations: the expected 2.3 kb length and about 4.6 kb unresolved self-complementary forms.

“When reads from the ssCB6-Egfp vector packaged with AAV2 were mapped to the cisplasmid, the alignments showed that they predominantly spanned from ITR-to-ITR (Fig. 3c).”

• Also stated in: § AAV-GPseq reveals that AAV2.7m8-packaged vector genomes have a higher degree of truncated genomes than AAV2-packaged vectors · plain

  “However, we observed two populations, one at the expected length of 2.3 kb, and a second with lengths that were double the size (~4.6 kb) (Fig. 3d).”

May combine two claims.

Not all 7-mer peptide insertions necessarily lead to a higher frequency of truncated genomes, as MyoAAV did not appear to exhibit multiple vector genome bands by alkaline gels.

“Finally, our observations suggest that not all 7-mer peptide insertions necessarily lead to a higher frequency of truncated genomes, as MyoAAV did not appear to exhibit multiple vector genome bands by alkaline gels (Fig. 2b).”

May combine two claims.

The 7m8 modification itself does not cause truncation events; rather, truncation events are more prominent in AAV2.7m8-packaged vectors than in AAV2-packaged vectors.

“This observation suggests that the 7m8 modification itself does not cause the formation of truncation events; rather, truncation events for AAV2.7m8-packagedvectors were more prominent than with the AAV2-packaged vectors.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Intravitreal injections of rAAV have been reported to trigger inflammatory responses and can lead to systemic biodistribution of vectors to lymph nodes.

“However, IVT injections of rAAV have been reported to trigger inflammatory responses and can lead to the systemic biodistribution of vectors to lymph nodes [15].”

• cites [15] Timmers AM, Newmark JA, Turunen HT, Farivar T, Liu J, Song C, et al. Ocular inflammatory response to intravitreal injection of adeno-associated virus vector: relative contribution of genome and capsid. Hum Gene Ther. 2020;31:80–9. sourceNot checked — no full text of the source

May combine two claims.

The detection of oversized genomes may be due to library preparation bias and an artifact of read length normalization.

“Genomes greater than 5 kb were not observed in our alkaline gels (Fig. 1d), suggesting that the detection of these oversized genomes may be a result of library preparation bias and an artifact of the read length normalization process (see Materials and Methods).”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the present study, outcomes were only evaluated six weeks post-IVT injection.

“However, in the present study, we only evaluated outcomes six weeks post-IVT injection.”

Classified as probably not a substantial claim.

The conclusions were based on only three vector designs.

“Although our conclusions were based on only three vector designs”

Probably not a substantial claim: may not pass S3 (load-bearing).

Peptide insertions and other alterations may exacerbate the stability of the AAV2 capsid, favoring particles with smaller genomes.

“Such peptide insertions and other alterations may exacerbate the stability of the AAV2 capsid, favoring particles with smaller genomes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

AAV vectors have been widely reported to cause immunogenicity and inflammation in animals and humans through subretinal injection.

“As the most commonly used vectors for retinal gene therapy, AAV vectors have been widely reported for causing immunogenicity and inflammation in animals and humans through subretinal injection.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

The genome heterogeneity observed for the AAV2.7m8 capsid may be masking its full potential.

“The genome heterogeneity observed for the AAV2.7m8capsid may in fact be masking its full potential.”

• AssumptionRests on an assumption: The insertion disrupts the basic arginine residues implicated in AAV2's binding to HSPGs.

Probably not a substantial claim: may not pass S3 (load-bearing).

The AAV2.7m8 capsid was identified through selective enrichment following two rounds of in vivo screening for transducing photoreceptor cells.

“The capsid itself was identified through selective enrichment following two rounds of in vivo screening for transducing photoreceptor cells [18].”

• cites [18] Dalkara D, Byrne LC, Klimczak RR, Visel M, Yin L, Merigan WH, et al. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med. 2013;5:189ra76. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The peptide inserts may alter how the capsid protein interacts with Rep, resulting in the packaging of truncated genomes.

“Alternatively, the peptide inserts may alter how the capsid protein interacts with Rep, resulting in the packaging of truncated genomes.”