Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids
Open accessAbstract
Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction.
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TL;DR
This review assesses strategies for expressing transgenes that exceed the ~5 kb packaging capacity of recombinant AAV vectors, focusing on dual-vector designs (fragmented/oversized, overlapping, trans-splicing, and hybrid). It concludes that hybrid dual-vector systems are currently the most promising in most instances, but that no single system is optimal across all transgenes and tissues.
Key findings
- AAV packaging capacity is limited to roughly 5 kb: wtAAV genomes average ~4.7 kb, Hermonat et al. found ~5.6 kb as the maximum for recovery of significant infectious virus, and Dong et al. reported optimal packaging between 4.1 and 4.9 kb, with vectors >5.2 kb packaged very inefficiently. Claims that AAV5 could package up to 8.9 kb were not supported by later studies, which found such genomes truncated (packaged genomes never exceeded 5.2 kb).
- Because ~6% of human proteins have coding sequences exceeding 4 kb (median human protein is 375 amino acids), some therapeutic cDNAs (e.g., ABCA4 ~7.3 kb, dystrophin, dysferlin, CFTR, MYO7A, factor VIII) cannot fit in a single AAV vector.
- In a direct comparison (Ghosh et al.), hybrid vectors achieved LacZ expression equal to a single intact vector at MOI 20,000 vg/cell, and 71% at 1,000 vg/cell, outperforming overlapping (~25%) and trans-splicing (~50%) variants; in mouse tibialis anterior muscle, hybrid vectors yielded 81% of single-vector β-galactosidase activity versus 34% (overlapping) and 62% (trans-splicing).
- Hybrid systems are not universally superior: Zhang and Duan found a dual overlapping vector system gave ~3-fold higher muscle transduction than three hybrid systems, and Pryadkina et al. obtained similar results for dysferlin. Minimized AP bridging sequences (0.27/0.26 kb vs. 0.87 kb) expand payload by ~1.2 kb; a 77 bp F1 phage-derived homology region has also been used.
Methods
- Narrative literature review; no primary experimental data reported.
- Synthesizes published studies on AAV biology, packaging capacity limits, and dual/triple-vector strategies (fragmented/oversized, overlapping, trans-splicing, hybrid), including in vitro (e.g., HEK293, MO59K, C2C12) and in vivo (mouse models including mdx, mdx4cv, A/J, ABCA4−/−, sh1) comparisons.
- Discusses mechanistic hypotheses (homologous recombination, ITR-mediated concatamerization, Rad51C-dependent repair) and design variables (recombinogenic sequences, splice donor/acceptor sites, ITR heterology).
Limitations
- Stated by the paper: the literature on dual-vector efficacy is "broad and often conflicting"; the mechanism of transduction by fragmented and overlapping vectors is "as yet not fully understood"; efficiency of transgene expression from dual-vector systems has been inconsistent and is currently insufficient for many clinical applications; hybrid systems are not always the most efficient; and optimization must be done empirically per transgene and tissue.
- Evident from the text: as a narrative review it provides no new experimental data, and several cited comparisons rely on different transgenes, serotypes, cell/tissue types, and titration methods, which the authors note can themselves influence results.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
49 of 110 cited sources checked · 30 supported · 0 flagged · accuracy not yet measured
- Premisecitation§ Overlapping dual vectors · p. 4
Overexpression of MYO7a in HEK293 cells with AAV2 vectors was more efficient with a dual overlapping vector system than with a single oversized vector.
“Trapani et al. found the overexpression of MYO7a in HEK293 cells with AAV2 vectors to be more efficient when they used a dual overlapping vector system as opposed to a single oversized vector, 46”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
- Premisecitation§ Overlapping dual vectors · p. 5
The method of vector titration can influence the results obtained when comparing two different strategies to increase AAV payload.
“even such seemingly arcane technical details as the method of vector titration appear to be able to influence the results obtained when comparing two different strategies to increase AAV payload. 46”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
The Materials and Methods section describes how vector titers were measured, and the Results compare strategies for delivering large genes. Neither passage says that the method of titration influences the comparison. The Discussion suggests other possible reasons for differences between studies, including vector design and purification, but does not identify titration method as one. (LLM, conf=0.76)
Experimental — accuracy not yet measured
- Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 3
Transduction of airway epithelial cells with an rAAV vector containing a truncated CFTR expression cassette was sufficient to reverse, at least partially, the cystic fibrosis chloride transport defect in these cells.
“Transduction of airway epithelial cells with an rAAV vector containing this expression cassette was sufficient to reverse, at least partially, the cystic fibrosis chloride transport defect in these cells. 35”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [35] source
- Premisecitation§ EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 2
The median length of a human protein is 375 amino acids.
“the median length of a human protein is only 375 amino acids. 27”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [27] source
The Results section (‘Protein lengths across proteomes’; page unavailable) reports a median of 361 amino acids for the pooled eukaryotic proteins, not a human-specific median. The Methods section (‘Proteomes’; page unavailable) says human proteins were included, but searches for ‘375’ and ‘Homo sapiens Table 2 median’ found no passage in our copy giving the claimed human value. (LLM, conf=0.72)
Experimental — accuracy not yet measured
- Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 3
A similar dual-vector strategy demonstrated phenotypic correction of FVIII deficiency in a mouse model of hemophilia A.
“Hu and Lipshutz used a similar dual-vector strategy to demonstrate phenotypic correction of FVIII deficiency in a mouse model of hemophilia A. 39”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [39] source
- Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 4
In mdx mice, gene transfer with AAV vectors encoding microdystrophin provides significant, although not optimal, therapeutic benefit.
“in mdx mice (a mouse model of Duchenne muscular dystrophy) gene transfer with AAV vectors encoding microdystrophin provides significant, although not optimal therapeutic benefit. 40-42”
Quote in the paperSubstantial claim1 of 3 sources checked - Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
During transduction, rAAV genomes form circular or linear (mostly) head-to-tail arrays of viral genomes.
“During transduction, rAAV genomes have been shown to form circular or linear (mostly) head-to-tail arrays of viral genomes, 14,50,51”
Quote in the paperSubstantial claim2 of 3 sources checked- cites [14] Duan D, 1998, J Virol source
The Results and Discussion report head-to-tail circular rAAV concatemers in infected muscle tissue. The Results say that linear integrated concatemers had previously been hypothesized, whereas this study's data suggest the persistent genomes are circular and episomal. The claim's assertion that linear arrays have been shown to form is therefore not established by this paper. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [50] source
The Abstract reports circular rAAV intermediates after transduction and describes monomer and multimer head-to-tail conformations. The paper does not establish that linear arrays formed during transduction are mostly head-to-tail; its statement about the most abundant head-to-tail form is limited to a model of circular genomes. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [51] source
- Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 3
The CMV promoter can be shortened to 173 bp without apparent loss in expression levels, at least in certain cell types.
“it has been reported that the CMV promoter can be shortened to 173 bp without apparent loss in expression levels-at least in certain cell types. 35”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [35] source
- Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
AAV vectors with two complementary ITRs can form circular concatamers.
“AAV vectors with two complementary ITRs can form circular concatamers. 13-16”
Quote in the paperSubstantial claim2 of 4 sources checked- cites [13] source
- cites [14] Duan D, 1998, J Virol source
- cites [15] source
- cites [16] source
The Discussion (page unavailable) says recombinant AAV derivatives can assume circular multimeric structures, supporting the circular-concatamer part of the claim. It also suggests that ITRs contribute to circle formation, but does not establish the specific condition of two complementary ITRs. The concatameric arrays described in the Results were linear products of rolling-circle amplification, not circular vector concatamers. (LLM, conf=0.83)
Experimental — accuracy not yet measured
- Premisecitation§ Overlapping dual vectors · p. 4
Overexpression of dysferlin by intramuscular injection of AAV8 encoding dysferlin from either a single oversized vector or two overlapping vectors showed approximately 10-fold higher expression of dysferlin with the dual-vector system.
“overexpression of dysferlin by intramuscular injection of AAV8 encoding dysferlin from either a single oversized vector or from two overlapping vectors showed a * 10-fold higher expression of dysferlin with the dual-vector system. 45”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [45] source
The Results section on intramuscular injection reports higher dysferlin expression with the overlapping dual-vector Set 3 than with the oversized fAAV vector. It gives the increase as approximately 4–10-fold relative to Batch 2 fAAV, so the citing sentence’s approximately 10-fold figure omits the lower end of the reported range. (LLM, conf=0.87)
Experimental — accuracy not yet measured
- Interpretationreasoning§ PACKAGING CAPACITY OF VIRUSES OF THE PARVOVIRIDAE FAMILY · p. 2
The maximum packaging capacity of members of the Parvoviridae family is approximately 4.5-5.5 kb.
“This suggests that the maximum packaging capacity of members of the Parvoviridae family is approximately 4.5-5.5 kb.”
Quote in the paperSubstantial claim3 of 8 sources checked- cites [8] source
The Introduction reports that AAV-1 DNA in a particle should not exceed 1.7 × 10⁶ daltons, and the paper describes one DNA strand per AAV virion. These passages support an approximate genome size for AAV, but they do not establish a 4.5–5.5 kb maximum packaging capacity for the Parvoviridae family. No page number is supplied in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [18] de la Maza LM, 1980, J Biol Chem source
- cites [19] Srivastava A, 1983, J Virol source
The available passage reports that the adeno-associated virus 2 genome is 4,675 nucleotides long, within the claimed 4.5–5.5 kb range. It does not establish a maximum packaging capacity for AAV2 or for the Parvoviridae family; searches for packaging capacity and encapsidation found no such passage. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [20] source
- cites [21] source
The Introduction identifies goose parvovirus as a member of Parvoviridae and gives its genome length as approximately 5,106 nt; the Results and Discussion give 5,050 nt for the swan isolate. Those lengths fall within the claimed range, but the paper does not establish a maximum packaging capacity for the family. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [22] Wong FC, 1985, Can J Comp Med
- cites [23] source
- cites [24] source
- Premisecitation§ Overlapping dual vectors · p. 4
Expression of MYO7A in photoreceptors was stronger with a dual overlapping vector system compared with a single oversized vector.
“Dyka et al. demonstrated that the expression of MYO7A in photoreceptors was stronger with a dual overlapping vector system when compared with a single oversized vector. 47”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [47] source
The Results section on MYO7A expression in mouse retina reports expression in photoreceptors with both the simple-overlap dual vector and the single oversized fAAV vector. It also reports that simple-overlap MYO7A was detectable by Western blot while fAAV MYO7A was not, but that comparison used whole-retina protein, not a photoreceptor-specific measure of expression strength. No page number is available in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measured
- Premisecitation§ Overlapping dual vectors · p. 5
A study found dual overlapping vectors less efficacious than a single vector with a genome of approximately 7.9 kb for delivering MYO7A to mouse retinas.
“a study investigating the efficacy of AAV2 and AAV5 vectors to deliver MYO7A, mutations in which cause Usher syndrome 1b, to mouse retinas found the dual overlapping vectors to be less efficacious than a single vector with a genome of * 7.9 kb in size. 48”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [48] source
The Introduction and Discussion report that overlapping dual AAV2 vectors were substantially less effective than single AAV2 or AAV5 vectors at delivering functional MYO7A to retinas of Myo7a-mutant mice. The Results describe the single vector’s 6.7-kb MYO7A cDNA and say the construct exceeds nominal AAV carrying capacity by about 2 kb, but our copy does not establish the claimed approximately 7.9-kb genome size. (LLM, conf=0.85)
Experimental — accuracy not yet measured
- Premisecitation§ Overlapping dual vectors · p. 4
Two overlapping AAV vectors encoding a dysferlin expression cassette produced high levels of muscle transduction.
“the authors found high levels of muscle transduction with two overlapping AAV vectors encoding a dysferlin expression cassette ( dysferlin has a cDNA size of 6.5 kb and the overlap between the two vectors was * 1kb). 44”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [44] source
- Premisecitation§ Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids · p. 1
rAAV genome-packaging capacity is approximately 5 kb.
“One limitation of rAAVs is that their genome-packaging capacity is only * 5kb.”
- Also stated in: § EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 3 · plain
“In fact, these reports confirmed earlier studies that the packaging capacity of AAV is roughly 5 kb.”
Quote in the paperSubstantial claim1 of 3 sources checked - Also stated in: § EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 3 · plain
- Premisecitation§ Overlapping dual vectors · p. 5
The nature of the transgene and tissue/cell type can influence the efficiency of dual vectors.
“it has been reported that the nature of the transgene 47 and tissue/cell type (possibly because of differences in DNA repair pathways) 45,46 can influence the efficiency of dual vectors.”
Quote in the paperNot a substantial claim3 of 3 sources checked - Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 4
The microdystrophin variant can be packaged in a single AAV vector.
“the so-called microdystrophin variant can be packaged in a single AAV vector”
Quote in the paperSubstantial claim1 of 3 sources checked - Interpretationcitation§ Hybrid dual-vector systems · p. 9
There is no one-fits-all dual-vector system.
“But they also demonstrate that there is no one-fits-all dual-vector system, and that the nature of the artificial recombinogenic regions and the tissue being targeted can drastically influence the efficiency of transduction with such hybrid split AAV vector systems. 46”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
- Interpretationcitation§ Hybrid dual-vector systems · p. 9
The choice of the best dual-vector design can depend significantly on the nature of the therapeutic protein.
“These results provide further evidence that the choice of the best dual-vector design can depend significantly on the nature of the therapeutic protein.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [68] source
The Discussion reports that the best-performing dual-vector set for mini-dystrophin depended on the gene-splitting site and the sequence used for recombination; the Results also show significantly higher transduction with set IV. But the study compares designs for one therapeutic protein, not different proteins, so it does not directly establish that the preferred design depends on the nature of the protein. (LLM, conf=0.82)
Experimental — accuracy not yet measured
- Premisecitation§ Hybrid dual-vector systems · p. 9
Vectors using the hybrid AP system expressed very poorly when compared with overlapping, trans-splicing, and hybrid-AK vectors.
“But, interestingly, they also found that the vectors using the hybrid AP system expressed very poorly when compared with overlapping, trans-splicing, and hybrid-AK vectors. 46”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
- Premisecitation§ Hybrid dual-vector systems · p. 9
In the mdx4cv mouse model of Duchenne muscular dystrophy, muscle transduction was significantly greater when the dual overlapping vector system was used, with an expression level approximately three times higher than with vectors based on the hybrid approach.
“Interestingly, they found that muscle transduction in the mdx4cv mouse model of Duchenne muscular dystrophy was significantly greater when the dual overlapping vector system was used-with an expression level approximately three times higher than with vectors based on the hybrid approach.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [68] source
Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measured
- Premisecitation§ Hybrid dual-vector systems · p. 8
When three minimal AP HR constructs were compared to the phage F1 HR constructs in HEK293 cells, one of the shortened AP HR vectors (AP1) showed slightly higher expression levels compared with the two other vector systems.
“When the authors compared three minimal AP HR constructs to the phage F1 HR constructs in HEK293 cells, one of the shortened AP HR vectors (AP1) showed slightly higher expression levels compared with the two other vector systems. 60”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [60] source
The Results section (page not provided) compares three AP-derived regions (AP, AP1 and AP2) with the F1-phage-derived AK region in HEK293 cells. It reports that both AP1 and AP2 produced slightly higher expression than AK, while all three outperformed AP. It does not establish that AP1 expressed more than AP2, as the claim’s comparison with the other systems implies. (LLM, conf=0.88)
Experimental — accuracy not yet measured
- Premisecitation§ Hybrid dual-vector systems · p. 8
The homologous regions on both vectors could be reduced to 0.27 and 0.26 kb, respectively, without apparent loss of transduction efficiency when compared with the original hybrid vectors (with homology regions of 0.87 kb).
“Using this approach, the authors were able to demonstrate that the homologous regions on both vectors could be reduced to 0.27 and 0.26 kb, respectively, without apparent loss of transduction efficiency when compared with the original hybrid vectors (with homology regions of 0.87 kb).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [67] source
The results accompanying Figures 3B and 4 report that hybrid-vector sets using 0.27-kb or 0.26-kb bridging sequences performed at least as well as the original 0.87-kb set. But these were separate sets, each using its stated bridging sequence; the paper does not report a pair with 0.27 kb on one vector and 0.26 kb on the other. Page numbers are unavailable in our copy. (LLM, conf=0.85)
Experimental — accuracy not yet measured
- Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
Auricchio and colleagues did not observe a significant increase in full-length ABCA4 production when comparing homologous and heterologous ITR vector pairs in HEK293 cells.
“Auricchio and colleagues did not observe a significant increase in fulllength ABCA4 production when they compared homologous and heterologous ITR vector pairs in HEK293 cells. 60”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [60] source
The Results (Figure 3B–D; page unavailable) compare homologous and heterologous ITR vectors in HEK293 cells and report no increase in full-length protein when cells were dosed by transgene titre. But when dosed by ITR2 titre, heterologous vectors produced higher levels of both full-length and truncated protein. The Discussion says there was no significant increase in the ratio of full-length to truncated protein, which is narrower than the claim's unqualified statement about full-length ABCA4 production. (LLM, conf=0.87)
Experimental — accuracy not yet measured
- Premisecitation§ Hybrid dual-vector systems · p. 8
In vivo, ABCA4 expression in photoreceptor cells occurred more frequently in mice injected subretinally with F1 HR vectors when compared with mice injected with AP HR vectors.
“On the other hand, in vivo, ABCA4 expression in photoreceptor cells occurred more frequently in mice injected subretinally with F1 HR vectors when compared with mice injected with AP HR vectors. 60”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [60] source
The Results section on mouse photoreceptor transduction (page unavailable) reports more frequent efficient ABCA4 expression after subretinal injection with AK vectors, whose homology region derives from F1 phage, than with AP1 or AP2 vectors. It does not report an in vivo comparison with the distinct AP vectors named in the claim; those were tested in cells. (LLM, conf=0.86)
Experimental — accuracy not yet measured
- Premisecitation§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
A simple, dual overlap vector system outperformed three independent hybrid systems.
“For instance, Zhang and Duan demonstrated that a simple, dual overlap vector system outperformed three independent hybrid systems, 68 and Pryadkina et al. obtained similar results when they analyzed the efficiency of dysferlin overexpression obtainable with dual overlapping vector or hybrid vector systems. 45”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [68] source
- Premisecitation§ Hybrid dual-vector systems · p. 8
77 bp-long homology regions derived from the filamentous phage F1 were described by Auricchio and colleagues.
“Evenshorter, 77bp-long homology regions (HR), derived from the filamentous phage F1, were described by Auricchio and colleagues. 46”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
- Premisecitation§ Hybrid dual-vector systems · p. 9
Inclusion of an in-frame degron after the splice-donor site (for the 5′ vector) prevents the accumulation of such fragments.
“A potential solution to this challenge was provided by Auricchio and colleagues, who showed that the inclusion of an in-frame degron after the splice-donor site (for the 5 ¢ vector) prevents the accumulation of such fragments. 60”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [60] source
- Interpretationcitation§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
Hybrid systems are not always the most efficient vector systems available.
“It needs to be pointed out, however, that hybrid systems are not always the most efficient vector systems available.”
Quote in the paperNot a substantial claim2 of 2 sources checked - Premisecitation§ Hybrid dual-vector systems · p. 9
In vitro transduction with vectors with oversized genomes did not result in efficient transgene expression.
“In their study, Trapani et al. demonstrated that in vitro transduction with vectors with oversized genomes did not result in efficient transgene expression. 46”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
The Results section (page unavailable) reports that oversized AAV vectors produced transgene expression in vitro, but were less efficient than several dual-AAV approaches. It does not establish that expression from oversized vectors was inefficient in absolute terms; one dual-AAV approach produced even lower expression. (LLM, conf=0.85)
Experimental — accuracy not yet measured
- Interpretationcitation§ Hybrid dual-vector systems · p. 8
These reduced bridging sequences allow for the expansion of the transgene payload by approximately 1.2 kb in a dual hybrid AAV vector system.
“Thus, these reduced bridging sequences allow for the expansion of the transgene payload by * 1.2 kb in a dual hybrid AAV vector system. 67”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [67] source
- Premisecitation§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
Pryadkina et al. obtained similar results when they analyzed the efficiency of dysferlin overexpression obtainable with dual overlapping vector or hybrid vector systems.
“For instance, Zhang and Duan demonstrated that a simple, dual overlap vector system outperformed three independent hybrid systems, 68 and Pryadkina et al. obtained similar results when they analyzed the efficiency of dysferlin overexpression obtainable with dual overlapping vector or hybrid vector systems. 45”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [45] source
- Premisecitation§ EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 2
Approximately 6% of all human proteins have a coding sequence that exceeds 4 kb.
“approximately 6% of all human proteins have a coding sequence that exceeds 4 kb, 28”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [28] source
- Premisecitation§ Fragmented vectors · p. 4
No full-length viral genomes of approximately 8.2 kb could be detected by alkaline gel Southern blot.
“no full-length viral genomes of * 8.2 kb could be detected by alkaline gel Southern blot. 30”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
- Interpretationnone given§ Dual vectors: concatamers and trans-splicing · p. 6
For all dual-vector systems (and oversized fragmented vectors) to be successful, at minimum a cell must be co-infected by AAVs encoding the 5' and 3' part of the expression cassette.
“for all dual-vector systems (and oversized, i.e., fragmented vectors) to be successful-at a minimuma cell must be co-infected by AAVs encoding the 5 ¢ and 3 ¢ part of the expression cassette, respectively.”
Quote in the paperNot a substantial claim - Interpretationreasoning§ Dual vectors: concatamers and trans-splicing · p. 6
For trans-splicing and hybrid vector systems, coinfection of a cell by AAVs encoding the 5' and 3' portion must be followed by appropriate concatamerization of the two vectors.
“for trans-splicing vector systems, and for hybrid vector systems (in the absence of homologous recombination), coinfection of a cell by AAVs encoding the 5 ¢ and 3 ¢ portion must be followed by appropriate concatamerization of the two vectors.”
Quote in the paperSubstantial claim - Premisecitation§ Overlapping dual vectors · p. 4
In mice, the best overlapping vector pair yielded almost identical transduction of the airway epithelium compared with the single-genome virus.
“In vivo , the best overlapping vector pair yielded almost identical transduction of the airway epithelium when compared with the single-genome virus.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [43] source
- Premisecitation§ PACKAGING CAPACITY OF VIRUSES OF THE PARVOVIRIDAE FAMILY · p. 2
rAAVs can package genomes ranging from 2 to 5.2 kb in size.
“Dong et al. 26 reported that rAAVs can package genomes ranging from 2 to 5.2 kb in size.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26] source
- Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 3
Co-injection of two AAV vectors encoding a truncated but functional FVIII heavy chain and the FVIII light chain into C57/BL6 mice resulted in production of therapeutic levels of biologically active FVIII.
“co-injection of the two vectors into C57/BL6 mice resulted in the production of therapeutic levels of biologically active FVIII.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [38] source
- Premisecitation§ Overlapping dual vectors · p. 4
Expression of mini-dystrophin was observed in striated muscle of mdx mice when rAAV6 was given intravenously.
“expression of mini-dystrophin was observed in striated muscled of mdx-mice when rAAV6 was given intravenously.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [42] source
- Premisecitation§ EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 3
The oversized AAV virus stock was in fact a mixture of partially packaged viruses.
“further molecular characterization found that the oversized virus stock was in fact a mixture of partially packaged viruses.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
- Premisecitation§ EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 3
Packaged AAV vector genomes never exceeded 5.2 kb in length.
“Wu et al., 32 who found that packaged vector genomes never exceeded 5.2 kb in length”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [32] source
- Premisecitation§ PACKAGING CAPACITY OF VIRUSES OF THE PARVOVIRIDAE FAMILY · p. 2
The optimum packaging size for AAV genomes is between 4.1 and 4.9 kb.
“The optimum packaging size for genomes was determined to be between 4.1 and 4.9 kb”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26] source
- Premisecitation§ Fragmented vectors · p. 4
In a slot blot with probes against AP and LacZ open reading frames, both probes hybridized to viral DNA from purified rAAV.
“in a slot blot with probes directed against the AP and the LacZ open reading frames (ORFs), both probes hybridized to viral DNA from purified rAAV.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
- Premisecitation§ EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 3
Plasmid-encoded vectors exceeding 5.2 kb existed as a mixture of genomes that were heterogeneous in size.
“plasmid encoded vectors exceeding 5.2 kb existed as a mixture of genomes that were heterogeneous in size.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [32] source
- Interpretationnone given§ Dual vectors: concatamers and trans-splicing · p. 6
For overlapping vector systems, nuclear import and uncoating must be followed by homologous recombination of the 5' and 3' parts of the expression cassette.
“For overlapping vector systems, nuclear import and uncoating must then be followed by homologous recombination of the 5 ¢ and 3 ¢ parts of the expression cassette.”
Quote in the paperSubstantial claim - Premisecitation§ Overlapping dual vectors · p. 4
The overlap method was used successfully with a split mini-dystrophin expression cassette with a combined length of approximately 7.3 kb and a short 372 bp overlap region.
“the overlap method was also used successfully with a split mini-dystrophin expression cassette with a combined length of approximately 7.3 kb and a short, 372 bp overlap region. 42”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [42] source
- Premisecitation§ Overlapping dual vectors · p. 4
Transduction with overlapping vector pairs was detected only with vector pairs that shared either 440 or 1000-base regions of homology but not with vector pairs without homology.
“they could detect transduction only with vector pairs that shared either 440or 1000-base regions of homology but not with vector pairs without homology. 43”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [43] source
- Premisecitation§ SHRINKING THE SIZE OF THE TRANSGENE CASSETTE BY MINIMIZING THE SIZE OF REGULATORY ELEMENTS AND SPLITTING OR TRUNCATING THE THERAPEUTIC PROTEIN · p. 3
The 32-bp-long polyadenylation signal of soluble neurophilin 1 is equally efficient in mRNA polyadenylation compared with the late SV40 polyadenylation signal.
“the 32-bp-long polyadenylation of soluble neurophilin 1 is equally efficient in mRNA polyadenylation when compared with the late SV40 polyadenylation signal, 36”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [36] source
- Premisecitation§ Overlapping dual vectors · p. 4
Simultaneous infection of cells with two vectors containing an overlapping fragment of 440 or 1000 bases of the AP gene could successfully transduce HT-1080 cells, although only at 1-2% efficiency of a vector that expressed AP from a single vector genome.
“simultaneous infection of cells with two vectors containing an overlapping fragment of 440 or 1000 bases of the AP gene could successfully transduce HT-1080 cells, although only at 1-2% efficiency of a vector that expressed AP from a single vector genome. 43”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [43] source
- Interpretationreasoning§ Hybrid dual-vector systems · p. 9
Hybrid dual AAV vector systems are a promising platform to express large genes with AAV vectors.
“Taken together, these results provided further evidence that hybrid dual AAV vector systems are a promising platform to express large genes with AAV vectors.”
Quote in the paperSubstantial claim - Interpretationreasoning§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
Overall, if carefully optimized, the hybrid vector approach, which combines the overlapping sequence and trans-splicing methodologies, appears to be, at least for now, the most promising approach in most instances.
“Nonetheless, overall-if carefully optimized-the hybrid vector approach, which combines the overlapping sequence and trans-splicing methodologies, appears to be, at least for now, the most promising approach in most instances.”
Quote in the paperSubstantial claim - Interpretationnone given§ Hybrid dual-vector systems · p. 8
Hybrid AAV vectors are not always the best available vector system to overexpress genes that exceed the packaging capacity of a single AAV capsid.
“These promising results notwithstanding, it needs to be pointed out that hybrid AAV vectors are not always the best available vector system to overexpress genes that exceed the packaging capacity of a single AAV capsid (see below).”
Quote in the paperNot a substantial claim - Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
A/J mice do not express functional dysferlin.
“A/J mice, which do not express functional dysferlin, 64 were injected intramuscularly with dual rAAV vectors, encoding the 5 ¢ and 3 ¢ parts of the dysferlin expression cassette, respectively.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [64] source
- Premisecitation§ Hybrid dual-vector systems · p. 8
The size of the AP sequence (0.87 kb within each vector) was a burden on the packaging capacity of the two vectors, effectively reducing the combined genome capacity of the dual-vector system from 10 to 8.2 kb.
“However, the size of the AP sequence (0.87 kb within each vector) was a burden on the packaging capacity of the two vectors, effectively reducing the combined genome capacity of the dual-vector system from 10 to 8.2 kb. 65”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [65] source
- Premisecitation§ Hybrid dual-vector systems · p. 7
The 872 bp AP fragment is highly recombinogenic in the overlapping vector system.
“The choice of the 872 bp AP fragment was based on earlier work that showed that this AP fragment is highly recombinogenic in the overlapping vector system. 66”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [66] source
- Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
Intramuscular injection of a vector pair with heterologous ITRs resulted in a 3-6-fold higher expression level compared with a homologous vector pair.
“intramuscular injection of a vector pair with heterologous ITRs resulted in a 3-6-fold higher expression level when compared with a homologous vector pair. 49,59”
Quote in the paperSubstantial claim0 of 2 sources checked - Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
In A/J mice injected intramuscularly with dual rAAV vectors encoding the 5′ and 3′ parts of the dysferlin expression cassette, successful trans-splicing resulted in expression of full-length dysferlin.
“As expected, successful trans-splicing resulted in the expression of full-length dysferlin, leading to improved membrane repair capacity, muscle histology, and muscle function. 63”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [63] source
- Interpretationreasoning§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
The choice of what the best vector system is can vary drastically depending on the transgene and the cell type/tissue targeted by the AAV vectors.
“Maybe the firmest conclusion that can be drawn from the vast trove of available literature is that there is no one ideal system, and that the choice of what the best vector system is can vary drastically depending on the transgene and the cell type/ tissue targeted by the AAV vectors.”
Quote in the paperSubstantial claim - Premisecitation§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
Koo et al. created a triple-vector system based on the trans-splicing approach that was capable of delivering the full-length 11.1 kb dystrophin genome via three vectors.
“For example, Koo et al. 70 created a triple-vector system based on the trans-splicing approach that was capableofdeliveringthefull-length11.1kb dystrophin genome via three vectors.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [70] source
- Interpretationreasoning§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
So far, the efficiency of transgene expression from dual-vector systems has been inconsistent.
“So far, however, the efficiency of transgene expression from dual-vector systems has been inconsistent.”
Quote in the paperSubstantial claim - Interpretationreasoning§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10
At present, for many clinical gene therapy applications, the efficiency of expression from the available dual-vector systems is insufficient.
“At present, for many clinical gene therapy applications, the efficiency of expression from the available dual-vector systems is insufficient.”
Quote in the paperSubstantial claim - Premisecitation§ Hybrid dual-vector systems · p. 7
Even when only 1,000 vector genomes per cell were used, the transduction efficiency of the hybrid vectors reached 71% of the single vector with a genome size fitting into a single AAV capsid.
“Even when only 1,000 vector genomes per cell were used, the transduction efficiency reached 71% of the single vector with a genome size fitting into a single AAV capsid. 65”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [65] source
- Interpretationreasoning§ Dual vectors: concatamers and trans-splicing · p. 6
The formation of correct concatamers of two or more AAV genomes likely increases as the copy number of AAV genomes in the nucleus rises.
“it is likely that the formation of ''correct'' concatamers of two, or more, AAV genomes increases as the copy number of AAV genomes in the nucleus rises.”
Quote in the paperSubstantial claim - Premisecitation§ Hybrid dual-vector systems · p. 7
When MO59K cells were transduced with the hybrid vectors at a multiplicity of infection of 20,000 viral genomes per cell, LacZ expression from the hybrid vectors was equal to that of the single intact vector.
“When MO59K cells were transduced with the hybrid vectors at a multiplicity of infection of 20,000 viral genomes per cell (10,000 vector genomes of each vector), the LacZ expression from the hybrid vectors was equal to that of the single intact vector.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [65] source
- Interpretationnone given§ Dual vectors: concatamers and trans-splicing · p. 6
Of the 10 possible two-vector linear concatamers, only one has the correct tail-to-head orientation.
“of the 10 possible two-vector linear concatamers, only one has the correct tail to head orientation.”
Quote in the paperSubstantial claim - Interpretationnone given§ Dual vectors: concatamers and trans-splicing · p. 6
A mandatory step for successful expression of proteins from two trans-splicing AAV vectors is that splicing of the pre-mRNA must occur across the ITR junction.
“A mandatory step for the successful expression of proteins from two trans-splicing AAV vectors is that splicing of the pre-mRNA must occur across the ITR junction (Fig. 1B).”
Quote in the paperSubstantial claim - Interpretationcitation§ Hybrid dual-vector systems · p. 9
The homologous recombination efficiency of particular artificial recombinogenic sequences can vary drastically depending on the specific applications.
“Moreover, the homologous recombination efficiency of particular artificial recombinogenic sequences can vary drastically depending on the specific applications.”
Quote in the paperSubstantial claim - Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
With the optimal exon/intron/exon combination, approximately 80% of myofibers expressed mini-dystrophin.
“with the optimal exon/intron/exon combination approximately 80% of myofibers expressed mini-dystrophin. 61”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [61] source
- Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
Recent publications have shown greater success when researchers identified the optimal exon-splitting site in the design of dual trans-splicing vectors with other transgenes.
“Confirming these results, recent publications have shown greater success when the researchers identified the optimal exon-splitting site in the design of dual trans-splicing vectors with other transgenes.”
Quote in the paperNot a substantial claim - Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6
The nature of the splice site substantially influences transduction efficiencies.
“Intramuscular injection of AAV6 vectors with different exon/intron/ exon splitting sites demonstrated that the nature of the splice site substantially influences transduction efficiencies”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [61] source
- May combine two claims. 26
- Classified as probably not a substantial claim. 8
May combine two claims.
Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6rAAV genomes persist episomally as mostly circular concatamers in postmitotic cells, leading to long-term transgene expression.
“it is generally believed that rAAV genomes persist episomally as mostly circular concatamers 16 in postmitotic cells, leading to long-term transgene expression.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [16] source
The Discussion (page unavailable) reports that naturally acquired wild-type AAV persists mainly as circular episomes in human tissues and says this is consistent with circular episomal forms of recombinant AAV vectors. It does not establish that recombinant genomes are mostly circular concatamers in postmitotic cells or that this form leads to long-term transgene expression. The concatameric forms described in the Results are products of rolling-circle amplification, not evidence that the persisting genomes have that structure. (LLM, conf=0.86)
Experimental — accuracy not yet measured
May combine two claims.
Premisecitation§ EXPRESSING TRANSGENES THAT EXCEED THE PACKAGING CAPACITY OF AAV CAPSIDS · p. 3The finding that AAV5 capsids could package intact genomes of up to 8.9 kb in length was not supported by further studies; such genomes were truncated and the packaging capacity of AAV was still limited to genomes substantially smaller than 8.9 kb.
“However, the finding that AAV5 capsids could package intact genomes of up to 8.9 kb in length was not supported by further studies, which found that such genomes were truncated and that the packaging capacity of AAV was still limited to genomes substantially smaller than 8.9 kb. 30-32”
Quote in the paperSubstantial claim1 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation§ Overlapping dual vectors · p. 5Transduction by oversized vectors depends on the activity of the strand-annealing protein Rad51C but is independent of the catalytic subunit of DNA-dependent protein kinase.
“Samulski and colleagues reported that transduction by oversized vectors depends on the activity of the strand-annealing protein Rad51C but is independent of the catalytic subunit of DNA-dependent protein kinase. 34”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [34] source
May combine two claims.
Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6Trans-splicing vector-pair systems do not always outperform single oversized vectors; Hirsch et al. reported higher expression both in vitro and in vivo with a single oversized genome vector compared with a trans-splicing system.
“similar to dual overlapping vectors, trans-splicing vector-pair systems do not always outperform single oversized vectors. For instance, Hirsch et al. 34 reported higher expression both in vitro (C2C12 myoblasts and myotubules) and in vivo with a single oversized genome vector when compared with a transsplicing system. 34”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [34] source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6The trans-splicing method to expand the size of ORFs that can be successfully expressed using AAV vectors was originally developed in the laboratory of John Engelhardt and has since been utilized in several studies with varying success.
“The trans-splicing method to expand the size of ORFs that can be successfully expressed using AAV vectors was originally developed in the laboratory of John Engelhardt 52,53 and has since been utilized in several studies with varying success (e.g., refs. 53-57 ).”
Quote in the paperNot a substantial claim1 of 6 sources checkedMay combine two claims.
Premisecitation§ Hybrid dual-vector systems · p. 9Dual trans-splicing and hybrid-AK vectors successfully transduced mouse and pig photoreceptors and improved the phenotype of mouse models of STGD and USH1B.
“Consistent with their in vitro results, dual trans-splicing and hybrid-AK vectors successfully transduced mouse and pig photoreceptors and were found to improve the phenotype of mouse models of STGD and USH1B. 46”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
May combine two claims.
Premisecitation§ Hybrid dual-vector systems · p. 9Both Dyka et al. and Trapani et al. observed the expression of both full-length and smaller fragments (of defined size) of MYO7A and/or ABCA4 with the hybrid dual-vector system.
“Interestingly, however, both Dyka et al. 47 and Trapani et al. 46 observed the expression of both full-length and smaller fragments (of defined size) of MYO7A and or ABCA4 with the hybrid dual-vector system.”
Quote in the paperSubstantial claim2 of 2 sources checkedMay combine two claims.
Interpretationcitation§ Dual vectors: concatamers and trans-splicing · p. 6Circularization is an intramolecular event and is probably more efficient than intermolecular concatemerization.
“Because such a circularization is an intramolecular event, it is probably more efficient than intermolecular concatemerization. 34,49,58”
Quote in the paperSubstantial claim1 of 3 sources checkedMay combine two claims.
Interpretationcitation§ Hybrid dual-vector systems · p. 9The nature of the artificial recombinogenic regions and the tissue being targeted can drastically influence the efficiency of transduction with hybrid split AAV vector systems.
“But they also demonstrate that there is no one-fits-all dual-vector system, and that the nature of the artificial recombinogenic regions and the tissue being targeted can drastically influence the efficiency of transduction with such hybrid split AAV vector systems. 46”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [46] Trapani I, 2014, EMBO Mol Med source
May combine two claims.
Premisecitation§ Fragmented vectors · p. 4AAV5 vectors with supposedly 8.9 kb genomes resulted in expression of ABCA4, MYO7A, and CEP290 from genomes whose intact genome length would be 8.9, 8.1, and 8.9 kb, respectively.
“Nonetheless, AAV5vectorswith the supposedly 8.9kb large viral genomes clearly did result in expression of ABCA4, MYO7A, and CEP290 from genomes whose intact genome length would be 8.9, 8.1, and 8.9 kb in size, respectively.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [29] source
Probably not a substantial claim: may not pass S4 (based).
Interpretationreasoning§ Dual vectors: concatamers and trans-splicing · p. 6The efficiency of reconstituting a genome with a full-length expression cassette is likely dependent on the number of nuclear copies of vector genomes.
“the efficiency of reconstituting a genome with a full-length expression cassette is likely dependent on the number of nuclear copies of vector genomes.”
Quote in the paperNot a substantial claimMay combine two claims.
Premisecitation§ PACKAGING CAPACITY OF VIRUSES OF THE PARVOVIRIDAE FAMILY · p. 2AAV vectors larger than 5.2 kb are packaged very inefficiently, and their transduction efficiencies are dramatically reduced.
“vectors larger than 5.2 kb were found to be packaged very inefficiently, and transduction efficiencies of these oversized vectors were dramatically reduced. 26”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26] source
Classified as probably not a substantial claim.
Premisecitation§ PACKAGING CAPACITY OF VIRUSES OF THE PARVOVIRIDAE FAMILY · p. 2The maximum genome size that allowed recovery of significant amounts of infectious wild-type AAV was approximately 5.6 kb.
“Hermonat et al. concluded that the maximum genome size that allowed the recovery of significant amounts of infectious virus was approximately 5.6 kb. 25”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [25] source
Classified as probably not a substantial claim. May combine two claims.
Premisecitation§ Fragmented vectors · p. 4Southern blot hybridization revealed that purportedly oversized packaged genomes were in fact truncated, and the authors suggested that the truncated fragments could assemble to restore the full-length expression cassette.
“Southern blot hybridization revealed that purportedly oversized packaged genomes were in fact truncated, and the authors suggested that the truncated fragments could assemble thus restoring the full-length expression cassette.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
May combine two claims.
Premisecitation§ Fragmented vectors · p. 4Both AP expression and LacZ expression were detected in mdx muscle of adult mice injected with the oversized AAV vector preparations.
“both AP expression and LacZ expression were detected in mdx muscle of adult mice injected with such AAV vector preparations.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [30] source
May combine two claims.
Interpretationreasoning§ Overlapping dual vectors · p. 5The combination of AAV serotype and tissue/cell type might have a particularly profound effect on the efficiency of transduction with overlapping systems because a high level of co-transduction, preferably with multiple copies of each vector, is essential for efficient transgene overexpression.
“the combination of AAV serotype and tissue/cell type might have a particularly profound effect on the efficiency of transduction with overlapping systems because for dual -vector approaches to be successful, a high level of co-transduction-preferably with multiple copies of each vector-is essential for efficient transgene overexpression.”
Quote in the paperSubstantial claimMay combine two claims.
Premisecitation§ Overlapping dual vectors · p. 4Although overall levels of mini-dystrophin expression were lower than in wildtype muscles, there was an improvement in limb muscle physiological performance.
“Although the overall levels of minidystrophin expression were lower than in wildtype muscles, there was an improvement in limb muscle physiological performance. 42”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [42] source
May combine two claims.
Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6Vectors containing an artificial intron improved expression compared with trans-splicing vectors that contained a poor splice site, but expression was not as efficient as with the best trans-splicing vectors using natural dystrophin exon/intron sites.
“They could demonstrate improved expression with vectors containing this artificial intron when compared with trans-splicing vectors that contained a poor splice site; however, expression was not as efficient as with the best trans-splicing vectors using natural dystrophin exon/intron sites. 62”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [62] source
May combine two claims.
Premisecitation§ Dual vectors: concatamers and trans-splicing · p. 6The expression of full-length dysferlin led to improved membrane repair capacity, muscle histology, and muscle function.
“As expected, successful trans-splicing resulted in the expression of full-length dysferlin, leading to improved membrane repair capacity, muscle histology, and muscle function. 63”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [63] source
May combine two claims.
Premisecitation§ Hybrid dual-vector systems · p. 8Expression from the single vector with a regular size genome and the hybrid vectors was strong and widespread, whereas expression from the trans-splicing, and especially the overlapping vectors, was not as effective.
“Expression from the single vector with a ''regular size genome'' and the hybrid vectors was strong and widespread, whereas expression from the transsplicing, and especially the overlapping vectors, was not as effective.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [65] source
May combine two claims.
Premisecitation§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10Although full-length dystrophin could be detected with this three-vector system both in vitro and in vivo, the expression was very weak.
“Although full-length dystrophin could be detected with this three-vector systemboth in vitro and in vivo -the expression was very weak.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [70] source
Probably not a substantial claim: may not pass S2 (checkable).
Interpretationreasoning§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10There is no one ideal system.
“Maybe the firmest conclusion that can be drawn from the vast trove of available literature is that there is no one ideal system, and that the choice of what the best vector system is can vary drastically depending on the transgene and the cell type/ tissue targeted by the AAV vectors.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.
Premisenone given§ AUTHOR DISCLOSURE · p. 10Strictly speaking, trans-splicing and hybrid vector systems do not rely on trans-splicing of pre-mRNAs; instead, in both systems a full-length mRNA is generated whose manufactured intron is then removed by regular cis-splicing to join the two exons.
“{ Strictly speaking, trans-splicing and hybrid vector systems do not rely on trans-splicing of pre-mRNAs. Instead, in both systems a full-length mRNA is generated whose manufactured intron is then removed by regular cis -splicing to join the two exons.”
Quote in the paperNot a substantial claimMay combine two claims.
Premisecitation§ Hybrid dual-vector systems · p. 8When β-galactosidase activity from muscle tissue was quantified, the hybrid vectors yielded 81% of the activity of the single regular size genome vector, which was significantly greater than the β-galactosidase activity from overlapping (34%) or trans-splicing vectors (62%).
“When b -galactosidase activity from muscle tissue was quantified, the hybrid vectors yielded 81% of the activity of the single ''regular size genome'' vector, which was significantly greater than the b -galactosidase activity from overlapping (34%) or trans-splicing vectors (62%).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [65] source
May combine two claims.
Premisereasoning§ Hybrid dual-vector systems · p. 7Either pathway results in LacZ expression.
“Either pathway results in LacZ expression.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationnone given§ Hybrid dual-vector systems · p. 9Such fragments are likely nonfunctional and could be dominant-negative and/or toxic.
“Not only are such fragments likely nonfunctional, but they also could be dominant-negative and/or toxic.”
Quote in the paperNot a substantial claimMay combine two claims.
Premisereasoning§ Hybrid dual-vector systems · p. 7The two vectors of the hybrid system can lead to transcription of full-length mRNA either through AP-mediated homologous recombination or via ITR-mediated head-to-tail concatamerization.
“The two vectors of this hybrid system can lead to the transcription of full-length mRNA by one of two ways: either through AP -mediated homologous recombination or via ITR-mediated head-to-tail concatamerization (Fig. 2).”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationcitation§ Hybrid dual-vector systems · p. 7The hybrid vector outperformed both the overlapping and trans-splicing variants, which only expressed levels of approximately 25% and 50%, respectively, of the original construct.
“Thus, the hybrid vector outperformed both the overlapping and trans-splicing variants, which only expressed levels of approximately 25% and 50%, respectively, of the original construct.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [65] source
Probably not a substantial claim: may not pass S4 (based). May combine two claims.
Interpretationnone given§ CONCLUDING REMARKS AND FUTURE DIRECTIONS · p. 10The most important components of a dual hybrid vector system to be taken into account appear to be the choice of the recombinogenic region and the nature of splice donor and acceptor signals.
“In this context, the most important components of a dual hybrid vector system to be taken into account appear to be the choice of the recombinogenic region and the nature of splice donor and acceptor signals.”
Quote in the paperNot a substantial claim
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“provided another example of the successful use of an overlapping vector system to overexpress genes that exceed the packaging capacity of a single AAV vector.(44) In this study, the authors found high levels of muscle transduction with two overlapping AAV vectors encoding a dysferlin expression cassette (dysferlin has…”
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“More recently, Hu and Lipshutz used a similar dual-vector strategy to demonstrate phenotypic correction of FVIII deficiency in a mouse model of hemophilia A.(39) An example of successfully truncating a gene was reported by Ostedgaard et al.”
BackgroundEfficacy of Codelivery of Dual AAV2/5 Vectors in the Murine Retina and Hippocampus
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Adeno-Associated Virus (AAV) as a Vector for Gene Therapy
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“) [12], should be considered in these cases.”
BackgroundCRISPR-Cas9 DNA Base-Editing and Prime-Editing
Kantor, A., McClements, M. E., & MacLaren, R. E. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176240 · Cited by 490 · see citing works
“For many diseases, vector-mediated gene delivery is not feasible due to the cargo limit of viral vectors as well as the expression of multiple heterogeneous variants of the target gene [97,98].”
BackgroundSystemic AAV vectors for widespread and targeted gene delivery in rodents
Challis, R. C., Kumar, S. R., Chan, K. Y., Challis, C., Beadle, K., Jang, M. J., Kim, H. M., Rajendran, P. S., Tompkins, J. D., Shivkumar, K., et al. (2019) · Nature Protocols · doi:10.1038/s41596-018-0097-3 · Cited by 415 · see citing works
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“The correct reassembly of the full-length expression cassette in the nuclei of target cells results in the production of full-length mRNA and protein (Yan et al, 2000; Duan et al, 2001; Chamberlain et al, 2016).”
BackgroundStrategies for nonviral nanoparticle‐based delivery of CRISPR/Cas9 therapeutics
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“Full-length assembly can occur through recombination of homologous regions or/and trans-splicing (Chamberlain et al., 2016).”
BackgroundSpatiotemporal control of CRISPR/Cas9 gene editing
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“To overcome the size limitation challenge, dual vector systems have been proposed, where the larger genome would be recreated through recombination of the partial genomes.(32) In addition, the effort to develop cassettes for factor VIII (FVIII) expression serves as an object lesson in setting expectations for both eff…”
BackgroundViral Vectors for the in Vivo Delivery of CRISPR Components: Advances and Challenges
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“When both AAVs are used to transduce a cell, the full-length gene and/or protein are reconstituted (Chamberlain et al., 2016).”
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Ryanodine Receptor 1-Related Myopathies: Diagnostic and Therapeutic Approaches
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“RYR1 exceeds 159 kb and thus far supersedes the current packaging capacity for adenovirusmediated therapy (∼ 5 kb) [96].”
BackgroundTanenhaus, A., Stowe, T., Young, A., McLaughlin, J., Aeran, R., Lin, I. W., Li, J., Hosur, R., Chen, M., Leedy, J., et al. (2022) · Human Gene Therapy · doi:10.1089/hum.2022.037 · Cited by 124 · see citing works
“72 AAV-delivered transgenes form stable, nonreplicating circular episomal DNA, which is gradually lost in dividing cells, but persists in postmitotic cells, such as neurons, due to the very low cellular turnover.”
InfluentialCRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors
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“…recombinant AAV (rAAV) vectors has not been successfully extended, though strategies that enable episomal expression of transgenes that exceed the packaging capacity have been devised (Grieger and Samulski, 2005, Chamberlain et al., 2016, Nakai et al., 2000, Sun et al., 2000, Halbert et al., 2002).”
BackgroundAdeno-Associated Viral Vectors in Neuroscience Research
Haggerty, D. L., Grecco, G. G., Reeves, K. C., & Atwood, B. K. (2019) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2019.11.012 · Cited by 123 · see citing works
“Although AAV2 is the most widely used variant because it was the first serotype identified, numerous other serotypes with varying tropism exist, giving AAVs wide clinical applicability.(92,93,97) When combined, these characteristics have established AAVs as a leading vector choice for gene therapy studies with one of…”
BackgroundInfluentialChallenges for Therapeutic Applications of Opsin-Based Optogenetic Tools in Humans
Shen, Y., Campbell, R. E., Côté, D. C., & Paquet, M.-È. (2020) · Frontiers in Neural Circuits · doi:10.3389/fncir.2020.00041 · Cited by 113 · see citing works
“Existing serotypes have been ‘‘overpackaged’’ with mixed success and varying reproducibility and the consensus appears to be that AAV can be overpackaged by ∼10%, but with a concomitant reduction in both viral titers and in vivo transduction (Chamberlain et al., 2016).”
BackgroundSystemic delivery of adeno-associated viral vectors
Duan, D. (2016) · Current Opinion in Virology · doi:10.1016/j.coviro.2016.07.006 · Cited by 103 · see citing works
Current knowledge of SLC6A1-related neurodevelopmental disorders
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“, 2014) and (iii) the transporters’ relatively small size (599 aa) makes SLC6A1 a plausible candidate for viral-mediated gene therapy using adeno-associated virus vectors (Chamberlain et al., 2016).”
BackgroundAdeno-associated Virus (AAV) Dual Vector Strategies for Gene Therapy Encoding Large Transgenes.
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Ahmadi, S. E., Soleymani, M., Shahriyary, F., Amirzargar, M. R., Mahya, O., Fattahi, D., & Safa, M. (2023) · Cancer Gene Therapy · doi:10.1038/s41417-023-00597-z · Cited by 95 · see citing works
“This approach is carried out by a process called intein-mediated trans-splicing, which to some degree is similar to mRNA splicing [83, 84].”
BackgroundMcClements, M. E., Barnard, A. R., Singh, M. S., Charbel Issa, P., Jiang, Z., Radu, R. A., & MacLaren, R. E. (2019) · Human Gene Therapy · doi:10.1089/hum.2018.156 · Cited by 92 · see citing works
Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting
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“Consequently, there is great interest in increasing transgene size while still utilizing the AAV system and the use of a dual AAV vectors system has been extensively studied for different experimental and disease modalities (Chamberlain et al., 2016).”
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Cardiac gene therapy with adeno-associated virus-based vectors
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“On the contrary, for approximately onethird of the genes listed in Table 1, the size of the respective cDNA exceeds the packaging capacity of AAV, which is 5 kb (including ITRs and regulatory elements [11]).”
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Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid
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“, protein trans-splicing) [38,60] have been pursued; however, these still occur with lowered efficiency and a clear upper packaging threshold at 5.”
BackgroundDirected Evolution of CRISPR/Cas Systems for Precise Gene Editing
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Targeting Cancer with CRISPR/Cas9-Based Therapy
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Preclinical evaluation of the efficacy and safety of AAV1-hOTOF in mice and nonhuman primates
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“37 In the TS strategy, the fragment from splice donor (SD) to splice acceptor (SA) is spliced out by intracellular spliceosome after recombination and transcription of transgene, which generates a mature mRNA and translates functional protein.”
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The potential for targeted rewriting of epigenetic marks in COPD as a new therapeutic approach
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Dual and triple AAV delivery of large therapeutic gene sequences into the inner ear
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Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor
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Genetic Delivery and Gene Therapy in Pulmonary Hypertension
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Adeno-Associated Virus-Based Gene Therapy for Lifelong Correction of Genetic Disease
Brommel, C. M., Cooney, A. L., & Sinn, P. L. (2020) · Human Gene Therapy · doi:10.1089/hum.2020.138 · Cited by 42 · see citing works
“There are multiple strategies to circumvent the packaging limitation of AAV-based vector for delivery of Cas9 expression vectors (reviewed in Ref.(15)).”
BackgroundInfluentialAtaluren for the Treatment of Usher Syndrome 2A Caused by Nonsense Mutations
Samanta, A., Štingl, K., Kohl, S., Ries, J., Linnert, J., & Nagel‐Wolfrum, K. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20246274 · Cited by 38 · see citing works
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Dual-AAV delivery of large gene sequences to the inner ear
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Peptide ligands for the affinity purification of adeno‐associated viruses from HEK 293 cell lysates
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“AAV is a small, nonenveloped icosahedral virus, whose capsid can pack a linear single‐strand DNA (ssDNA) genome of up to about 5 kilobases (Chamberlain et al., 2016; Lai et al., 2010; Linden et al., 1996; Vasileva & Jessberger, 2005).”
BackgroundProtein Engineering Strategies to Expand CRISPR-Cas9 Applications
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“3 kb), which limits its efficient gene-based delivery via recombinant adenoassociated virus (rAAV) [80, 81].”
BackgroundGene delivery to the lungs: pulmonary gene therapy for cystic fibrosis
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Gene Transfer Therapy for Neurodevelopmental Disorders
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Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing
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“However, the large size of SpCas9 (1368 amino acids, (cid:1) 4.1 kb) limits its further applications for adeno-associated virus (AAV)- based in vivo gene therapy, as the limited cargo size of the AAV vector prevents co-packaging of both SpCas9 and sgRNA expression cassettes [10].”
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Splicing in the pathogenesis, diagnosis and treatment of ciliopathies.
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Clinical and Translational Landscape of Viral Gene Therapies
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Canine Models of Inherited Musculoskeletal and Neurodegenerative Diseases
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“A highly truncated, micro-dystrophin gene (<4 kb) (197, 198) or dual-AAV vectors used in combination to deliver a larger payload (199, 200).”
BackgroundThird-generation lentiviral gene therapy rescues function in a mouse model of Usher 1B
Schott, J. W., Huang, P., Morgan, M. A., Nelson‐Brantley, J., Koehler, A., Renslo, B., Büning, H., Warnecke, A., Schambach, A., & Staecker, H. (2023) · Molecular Therapy · doi:10.1016/j.ymthe.2023.10.018 · Cited by 25 · see citing works
“Although dual AAV vector systems, in which the transferred gene is split into two separate vectors that recombine in the target cell, 15 can allow the use of AAV vectors to target disorders with larger genes, this strategy may be associated with a loss in ef fi ciency.”
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Neurosurgical gene therapy for central nervous system diseases
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“Dual AAV vector approaches in which smaller transgenes can recombine to form a larger transgene are also being explored but have not made it to clinical trials for a neurological indication [86].”
BackgroundAAV Vector-Mediated Antibody Delivery (A-MAD) in the Central Nervous System
Marino, M., & Holt, M. G. (2022) · Frontiers in Neurology · doi:10.3389/fneur.2022.870799 · Cited by 24 · see citing works
“2 kb is inefficient, and unfavorably impacts on transduction (80).”
BackgroundRevolution of AAV in Drug Discovery: From Delivery System to Clinical Application
Yin, L., He, H., Zhang, H., Shang, Y., Fu, C., Wu, S., & Jin, T. (2025) · Journal of Medical Virology · doi:10.1002/jmv.70447 · Cited by 22 · see citing works
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“9 kb.(43) For example, CNS therapies for Pompe disease(44) and Parkinson’s disease(45) are based on delivery of relatively large genes such as GAA (2.”
BackgroundPhelps, M., & Balazs, A. B. (2021) · Frontiers in Immunology · doi:10.3389/fimmu.2021.734304 · Cited by 20 · see citing works
“Following transduction, these recombinant viral genomes form head-to-tail concatemers within the nucleus, where they persist as non-integrated episomal genomes (105, 106).”
BackgroundCRISPR genome engineering for retinal diseases
Kantor, A., McClements, M. E., Peddle, C. F., Fry, L. E., Salman, A., Cehajic‐Kapetanovic, J., Xue, K., & MacLaren, R. E. (2021) · Progress in molecular biology and translational science · doi:10.1016/bs.pmbts.2021.01.024 · Cited by 19 · see citing works
Viral Vectors, Engineered Cells and the CRISPR Revolution
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“10,11 Briefly, the overlapping dual vector approach requires that the basic elements of an AAV gene therapy transgene be split across two separate transgenes.”
Allen, D. G., Kalter, N., Rosenberg, M., & Hendel, A. (2023) · Pharmaceutics · doi:10.3390/pharmaceutics15051329 · Cited by 17 · see citing works
Chamberlain, K., Riyad, J. M., Garnett, T., Kohlbrenner, E., Mookerjee, A., Elmastour, F., Bénard, L., Chen, J., VandenDriessche, T., Chuah, M., et al. (2018) · Human Gene Therapy · doi:10.1089/hum.2017.188 · Cited by 17 · see citing works
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Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo
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Hughes, M. P., Nelvagal, H. R., Coombe-Tennant, O., Smith, D., Smith, C., Massaro, G., Poupon-Bejuit, L., Platt, F. M., & Rahim, A. A. (2023) · Cells · doi:10.3390/cells12121619 · Cited by 16 · see citing works
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“cases, as the CDS reconstitution is dependent on complex DNA repair pathways and can vary from different tissue/cell types, leading to diverse gene reconstitution rates [2,6].”
BackgroundStefanov, A., & Flannery, J. G. (2022) · Cold Spring Harbor Perspectives in Medicine · doi:10.1101/cshperspect.a041304 · Cited by 16 · see citing works
Current Advances in Adeno-Associated Virus-Mediated Gene Therapy to Prevent Acquired Hearing Loss
Wu, F., Sambamurti, K., & Sha, S. (2022) · Journal of the Association for Research in Otolaryngology · doi:10.1007/s10162-022-00866-y · Cited by 15 · see citing works
“However, this is not a major concern for most hearing-related applications, with a median human protein size of 375 amino acids [63], since the cDNA of about 78 % of the proteins associated with hereditary hearing loss are under the size limit of the”
BackgroundSehara, Y., Inaba, T., Urabe, T., Kurosaki, F., Urabe, M., Kaneko, N., Shimazaki, K., Kawai, K., & Mizukami, H. (2018) · European Journal of Neuroscience · doi:10.1111/ejn.14169 · Cited by 15 · see citing works
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Adeno-Associated Virus-Mediated Gene Therapy
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“tional components including: promoter, guide RNA (gRNA), and DNA template [34, 39, 40].”
BackgroundKumagai, S., Nakajima, T., & Muramatsu, S. (2024) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2024.2386339 · Cited by 11 · see citing works
“Since the large size of the SCN1A gene (cDNA ~6 kb) precludes packaging into AAV vectors, AAV-mediated gene replacement therapy is not feasible for SCN1A [92].”
Ibarra, C. A., da Silva, H. C. A., Voermans, N. C., et al. (2024) · British Journal of Anaesthesia · doi:10.1016/j.bja.2024.05.046 · Cited by 11 · see citing works
Gene Therapy: Will the Promise of Optimizing Lung Allografts Become Reality?
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“However, the use of dual overlapping vector strategies could circumvent this limit (61).”
BackgroundQuach, T., Stratton, H. J., Khanna, R., Mackey-Alfonso, S. E., Deems, N., Honnorat, J., Meyer, K., & Duchemin, A. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms232214188 · Cited by 11 · see citing works
P53 upregulation by USP7-engaging molecular glues
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“In addition, there is a limitation of the gene size that could be packaged into AAVs, precluding the expression of relatively large proteins 5 .”
BackgroundMessenger RNA as a personalized therapy: The moment of truth for rare metabolic diseases
Córdoba, K. M., Jericó, D., Sampedro, A., Jiang, L., Iraburu, M. J., Martini, P. G. V., Berraondo, P., Avila, M. A., & Fontanellas, A. (2022) · International review of cytology · doi:10.1016/bs.ircmb.2022.03.005 · Cited by 11 · see citing works
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A non-viral genome editing platform for site-specific insertion of large transgenes
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Delivery Systems of Retinoprotective Proteins in the Retina
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Advancement in CRISPR/Cas9 Technology to Better Understand and Treat Neurological Disorders
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Polymeric biomaterials in the treatment of posterior segment diseases
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Planet of the AAVs: The Spinal Cord Injury Episode
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“As a result of this the superinfection, in the event of the presence of helper viruses, cannot appear as rAAVs are not able to integrate into the wtAAV’s preferential integration and trigger long-term transgene expression in postmitotic tissues, most likely because they persist as episomes within the host cell [59].”
BackgroundGenes in pediatric pulmonary arterial hypertension and the most promising BMPR2 gene therapy
Dai, L., & Du, L. (2022) · Frontiers in Genetics · doi:10.3389/fgene.2022.961848 · Cited by 8 · see citing works
“The solution strategy that has been put forward to solve this problem is the use of new site-mutated AAVs.”
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Optogenetic therapy for retinal degenerative diseases: A review
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“113 For example, nanoparticles may not be able to penetrate the layers of tissue in the eye to reach the target cells and have high toxicity.”
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“…partial capsids in AAV formulations is that they do not help to improve overall transduction because they contain no therapeutic GOI and could inhibit AAVs containing GOI (e.g., via competition for target receptors, co-receptor recognition, or binding saturation) to reach the target cells (45–53).”
Viral Vectors for Gene Therapy
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Peek, J. L., Rosales, A., Qi, J., Feleke, L., Menshikh, A., Welch, R. C., Woodard, L. E., Asokan, A., & Wilson, M. H. (2025) · Molecular Therapy Advances · doi:10.1016/j.omta.2026.201736 · Cited by 6 · see citing works
Synthetic genomics for curing genetic diseases
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- Authors
- Chamberlain, K., Riyad, J. M., & Weber, T.
- Affiliations
- Icahn School of Medicine at Mount Sinai (US)
- Corresponding author
- Weber, Thomas
- Venue
- Human Gene Therapy Methods
- Publisher
- SAGE Publications
- Published
- 2016-02
- Volume
- 27
- Issue
- 1
- Pages
- 1-12
- ISSN
- 1946-6536 (ISSN-L)
- 1946-6544
- PMC
- PMC4761816
- OpenAlex
- W2280525353
- Semantic Scholar
- 2025d56e2cca1328dc2aed3bfe2dab7881704456
- Cited by
- 141
- References
- 74
- Citations by year
- 2026: 10 · 2025: 10 · 2024: 8 · 2023: 13 · 2022: 17 · 2021: 22 · 2020: 20 · 2019: 12 · 2018: 13 · 2017: 12 · 2016: 4
- FWCI
- 1.83
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Human Gene Therapy Methods (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Fondation Leducq (14CVD03)
- National Institutes of Health (HL112324)
- National Heart, Lung, and Blood Institute (P50HL 112324, HL117505, R01HL117505, HL112324)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- recombinant adeno-associated virus vectors, co-infection, transgene expression, gene therapy vectors, transduction efficiency
- MeSH terms
- Dependovirus, Animals, DNA, Viral, Genetic Vectors, Humans, Genetic Therapy, Gene Expression, Genome, Viral, Gene Transfer Techniques, Virus Assembly, Transgenes, Capsid Proteins, Genome Size
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