Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting

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

The three dual approaches have been used to express MYO7A and ABCA4 genes and showed effective in vitro protein expression.

“Furthermore, the three dual approaches (trans-splicing, overlapping or “hybrid” techniques) have been used to express the MYO7A and the ABCA4 genes (both with CDS over 6 kb in length and expressed in the retina) and showed effective in vitro protein expression (Dyka et al., 2014).”

• cites Dyka et al., 2014 sourcePartly supported

  The Results section (page not provided) reports full-length MYO7A protein expression in HEK293 cells using the dual-vector approaches. The Discussion mentions in vitro ABCA4 expression, but says the simple-overlap approach was highly inefficient relative to other platforms; it does not establish effective expression of ABCA4 with all three approaches. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

The difference between OL reconstitution in RPE cells and photoreceptors may be due to differences in overlap lengths and sequences.

“Even though others have detected OL reconstitution in RPE cells, but not photoreceptors, this may be due to differences in overlap lengths and sequences (Trapani et al., 2015).”

• cites Trapani et al., 2015 sourcePartly supported

  The Results section on recombinogenic regions (page unavailable) compares different homology sequences and reports differences in how frequently they yield ABCA4 expression in mouse photoreceptors. It also notes lower recombination-based vector efficiency in photoreceptors than in cell culture. It does not compare overlapping-vector reconstitution in RPE cells and photoreceptors or attribute such a difference to overlap length and sequence. (LLM, conf=0.77)

  Experimental — accuracy not yet measured

May combine two claims.

Both transcription and translation are strongly affected by exact DNA and mRNA sequences, respectively, as well as their cellular environments.

“Both transcription and translation are strongly affected by exact DNA and mRNA sequences, respectively, as well as their cellular environments (Sonenberg and Hinnebusch, 2009; Alpert et al., 2017).”

• cites Sonenberg and Hinnebusch, 2009 sourcePartly supported

  The Introduction (page not available in our copy) explains that mRNA sequences and structures affect translation initiation, while cellular conditions such as starvation or stress can alter translation. It does not establish the corresponding claim that DNA sequence and cellular environment affect transcription. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites Alpert et al., 2017 sourcePartly supported

  The Abstract states that DNA sequence, gene architecture, and chromatin regulate transcription elongation rates; the Introduction also discusses cell signaling and environmental cues as possible constraints. The paper does not address whether mRNA sequence or cellular environment strongly affects translation, so it supports the transcription part but not the full sentence attributed to it. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

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

Others have detected OL reconstitution in RPE cells, but not photoreceptors.

“Even though others have detected OL reconstitution in RPE cells, but not photoreceptors, this may be due to differences in overlap lengths and sequences (Trapani et al., 2015).”

• cites Trapani et al., 2015 sourceCould not verify

  The Results section reports photoreceptor expression from dual AAV hybrid vectors, while its RPE measurements concern lipofuscin. I searched for overlapping-vector (OL) reconstitution in RPE cells but not photoreceptors; the retrieved passages do not report that comparison. No page number was supplied for these passages. (LLM, conf=0.70)

  Experimental — accuracy not yet measured

May combine two claims.

AAV8 and Anc80L65 both transduce retinal cells with high efficiencies.

“Effect of serotype on vector reconstitution was studied by comparing AAV8 and Anc80L65, both of which transduce retinal cells with high efficiencies (Zinn et al., 2015).”

• cites Zinn et al., 2015 sourceSupported

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

Vector reconstitution has been reported in vitro in several cell lines, and in vivo in mouse liver, skeletal muscle, and retina.

“Vector reconstitution has been reported in vitro in several cell lines, and in vivo in mouse liver, skeletal muscle, and retina (Grieger and Samulski, 2005).”

• cites Grieger and Samulski, 2005 sourcePartly supported

  The introductory passage labelled Abstract describes split-vector systems that rely on recombination of two vector genomes within a cell, supporting the general idea of vector reconstitution. It does not report reconstitution in several cell lines or in mouse liver, skeletal muscle, or retina. The Results describe transduction of cultured cells, not those specific reconstitution findings; page numbers are unavailable in our copy. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

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

Different relative efficiencies of dual AAV vectors have been reported by others, especially regarding HB and TS.

“Interestingly, different relative efficiencies have been reported by others, especially regarding HB and TS (Dyka et al., 2014; Trapani et al., 2014).”

• cites Dyka et al., 2014 sourceSupported
• cites Trapani et al., 2014 sourceSupported

Classified as probably not a substantial claim.

Vector reconstitution in the retina has been detected for clinically relevant large transgenes, such as ABCA4 or MYO7A.

“Even though vector reconstitution in the retina has been detected for clinically relevant large transgenes, such as ABCA4 or MYO7A, due to lack of proper controls (e.g., using oversized monocistronic vectors) and accurate protein quantification methods, determining parameters for clinical approaches has been lacking (Dyka et al., 2014; Trapani et al., 2014, 2015).”

• cites Dyka et al., 2014 sourceSupported
• cites Trapani et al., 2014 sourceSupported
• cites Trapani et al., 2015 sourceSupported

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

An alternate method for expressing large proteins relying on intein protein sequences has been reported.

“An alternate method for expressing large proteins relying on intein protein sequences has also been reported (Li et al., 2008; Truong et al., 2015; Chew et al., 2016).”

• cites Li et al., 2008Not matched

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

• cites Truong et al., 2015 sourceSupported
• cites Chew et al., 2016 sourceSupported

May combine two claims.

In vitro, dual vector dose increases of 10-fold and 100-fold resulted in 17-fold and 102-fold increases in reporter transgene expression, respectively.

“We also studied whether the effect of dose of dual vector transduction correlates linearly with reporter transgene expression in vitro, and detected 17 and 102-fold increases in expression when dual vector dose was increased 10 and 100-fold to controls, respectively.”

May combine two claims.

In vitro, reconstitution efficiencies based on protein expression normalized to controls were 15.3% for HB, 17.4% for OL, and 10.3% for TS.

“When reconstitution efficiencies based on protein expression were quantified by normalizing lacZ expression levels to their respective controls, levels of 15.3, 17.4, and 10.3% were detected for HB, OL and TS, respectively.”

May combine two claims.

Functionality of dual vectors and controls was assessed in vitro, and protein expression from oversized reporter was similar to normal sized controls.

“Functionality of our dual vectors and controls was assessed in vitro, and showed that protein expression from oversized reporter was similar to normal sized controls.”

May combine two claims.

In vivo in mouse retina, dual vector reconstitution efficiencies were 9.07% of control for HB and 1.78% for TS.

“Reconstitution efficiencies were 9.07 and 1.78% of control for HB and TS, respectively (Figure 3A).”

• Also stated in: § Discussion · hedged

  “Our data indicates different relative efficiencies of reconstitution in vivo in mouse retina with levels compared to control, of 9.07% for HB, 1.78% for TS, and below detection for OL.”

May combine two claims.

In vivo, reconstitution efficiency was assessed to be ~10% based on protein activity, while mRNA amounts suggested ~60% efficiency.

“While reconstitution efficiency was assessed to be ~10% based on protein activity, mRNA amounts suggested ~60% efficiency.”

Probably not a substantial claim: may not pass S4 (based).

Each dual AAV approach will result in reconstitution of transcriptionally active Rainbow construct.

“Each approach will result in reconstitution of transcriptionally active Rainbow construct.”

May combine two claims.

The rate of rAAV genome reconstitution may be quantifiable, but resulting mRNA or protein levels are not, unless controls of identical sequences are used.

“Rate of rAAV genome reconstitution may be quantifiable, but resulting mRNA or protein levels are not, unless controls of identical sequences are used.”

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

Transfection experiments were done in triplicates with identical conditions to ensure equal transfection efficiencies.

“Transfection experiments were done in triplicates with identical conditions to ensure equal transfection efficiencies.”

Probably not a substantial claim: may not pass S4 (based).

The lacZ primer/probe assay was specifically designed to span the exact location where the lacZ gene is split into left and right constructs in the dual hybrid strategy.

“The lacZ primer/probe assay was specifically designed to span the exact location where the lacZ gene is split into left and right constructs in the dual hybrid strategy.”

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

The drastic decrease in OL efficiency in vivo may reflect differences in activities of pathways responsible for homologous recombination in vivo as compared to in vitro.

“The drastic decrease in OL efficiency may reflect differences in activities of pathways responsible of homologous recombination in vivo as compared to in vitro.”

May combine two claims.

Packaged AAV genomes never exceeded 5.2 kb in length, but overpackaging results in genome fragmentation.

“A study by Wu et al. (2010) showed that packaged AAV genomes never exceeded 5.2 kb in length, but overpackaging results in genome fragmentation.”

• cites Wu et al., 2010Not matched

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

May combine two claims.

The TS approach has several different ways that the left and right vectors could come together, which overall lowers the probability of correct concatamerization and consequently reconstitution efficiency.

“As we show in Figure 1E, there are several different ways that the left and right vectors from the TS approach could come together, which overall lowers the probability of correct concatamerization and consequently reconstitution efficiency.”

May combine two claims.

Correct reconstitution can be accurately quantified by measuring β-galactosidase with standard enzymatic assays because neither left nor right constructs contains a full lacZ coding sequence.

“As neither left nor right constructs contains a full lacZ coding sequence, correct reconstitution can be accurately quantified by measuring the amount of its protein product, β-galactosidase, with standard enzymatic assays.”

May combine two claims.

Dual AAV approaches can partially overcome the size limitation and deliver larger transgenes.

“Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes.”

• Also stated in: § Introduction · plain

  “It has been shown that the fragments will reassemble when transferred to a cell and produce a full-length protein (Yan et al., 2000, 2002; Duan et al., 2003; Hirsch et al., 2009).”

• cites Yan et al., 2000, 2002; Duan et al., 2003; Hirsch et al., 2009Not matched

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

Probably not a substantial claim: may not pass S4 (based).

Reconstitution based on uniform homologous sequences, such as in the hybrid approach, should result in equal efficiencies regardless of the transgene.

“In contrast, reconstitution based on uniform homologous sequences, such as in the hybrid approach, should result in equal efficiencies regardless of the transgene.”

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

In the retina, the dual approach was first reported by Reich et al. (2003) using trans-splicing to show β-galactosidase expression in vivo around 40% after AAV5 subretinal injection.

“In the retina, the dual approach was first reported by Reich et al. (2003) which used the trans-splice technique to show that β-galactosidase expression in vivo was around 40% after AAV5 subretinal injection (Reich et al., 2003).”

• cites Reich et al., 2003 sourceNot checked — no full text of the source

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

Original Rainbow sequence is anticipated to be restored only after OL reconstitution, whereas TS will have an intron containing an AAV ITR sequence, and HB will have an intron with AP or AP-ITR-AP sequence.

“Original Rainbow sequence is anticipated to be restored only after OL reconstitution, whereas TS will have an intron containing an AAV ITR sequence, and HB will have an intron with AP or AP-ITR-AP sequence (Figures 1D,E).”

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

All animal procedures were performed in accordance with protocols approved by institutional animal care and use committees at SERI and conformed to ARVO guidelines.

“All animal procedures were performed in accordance with protocols approved by the institutional animal care and use committees at SERI and conformed to the guidelines on the care and use of animals adopted by the Association for Research in Vision and Ophthalmology (Rockville, MD).”

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

The drastic differences in HB reconstitution within AP intron sequence seen between different studies may reflect undetected or unreported mutations between sequences, especially in the ITRs, indicating sensitivity of reconstitution to minor variations.

“While all designs seemingly use sequences originally reported by Ghosh et al. (2008), the drastic differences in HB reconstitution within AP intron sequence seen between the different studies, may reflect undetected or unreported mutations between sequences, especially in the ITRs, indicating sensitivity of reconstitution to minor variations (Ghosh et al., 2008).”

• cites Ghosh et al., 2008 sourceNot checked — no full text of the source

May combine two claims.

Regulatory sequences required to drive gene expression add to the final size and may render many genes too large for AAV packaging.

“Moreover, regulatory sequences that may be required to drive gene expression add to the final size and may render many genes too large for AAV packaging.”

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

For clinical purposes, dual AAV approaches may be feasible if sufficient protein expression levels to affect the disease phenotype can be achieved, regardless of the de facto efficiency of the reconstitution process.

“For clinical purposes, dual AAV approaches may be feasible if sufficient protein expression levels to affect the disease phenotype can be achieved, regardless of the de facto efficiency of the reconstitution process.”

Classified as probably not a substantial claim.

OL reconstitution depends on the recombinogenic capacity of the sequence, which is expected to be gene-dependent.

“and on the recombinogenic capacity of this sequence, which is expected to be gene-dependent.”

May combine two claims.

An enzymatic assay identifies reconstitution based on protein function, not just size, thus excluding potential incorrect reconstitution events.

“Further, an enzymatic assay identifies reconstitution based on protein function, not just size, thus excluding potential incorrect reconstitution events.”

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

In the intein approach, the transgene is divided into two parts, with the 5′ half expressing a C-terminal intein sequence and the 3′ half an N-terminal intein sequence.

“In this approach, the transgene is divided into two parts in which 5′ half of the gene will express a C-terminal intein sequence, and 3′ half an N-terminal intein sequence.”

Probably not a substantial claim: may not pass S2 (checkable).

As of now, dual AAV vector strategies may be of use in gene therapy modalities where even limited transgene expression has positive effects on disease phenotype.

“As of now, dual AAV vector strategies may be of use in gene therapy modalities where even limited transgene expression has positive effects on disease phenotype.”

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

Inteins are of bacterial origin and may be immunogenic.

“Finally, inteins are of bacterial origin, and as such may be immunogenic.”

May combine two claims.

In the intein approach, both vector halves need separate transcriptional regulators, and the added length of intein sequences diminishes gain of payload carrying capacity compared to genome reconstitution approaches.

“Also, as the two peptides need to be expressed, both vector halves will need their separate transcriptional regulators, and with the added length of the intein sequences, gain of payload carrying capacity is diminished as compared to genome reconstitution approaches.”

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

Reconstitution in the intein approach takes place on peptide level in a process termed protein splicing, where the intein sequences excise themselves and join the remaining peptides together.

“Reconstitution will therefore take place on peptide level in a process termed protein splicing, where the intein sequences excise themselves and join the remaining peptides together.”

Probably not a substantial claim: may not pass S4 (based). May combine two claims.

Proteins can be split by inteins only at certain sites, and the reconstituted protein may not reach proper conformation due to the two peptides folding separately.

“Further, proteins can be split by inteins only at certain sites, and the reconstituted protein may not reach proper conformation due to the two peptides folding separately.”

May combine two claims.

Co-transduction can also be achieved by intravitreal injection, which targets the retinal ganglion cells and inner retina, albeit at much lower efficiencies than by subretinal injections.

“However, co-transduction can also be achieved by intravitreal injection, which targets the retinal ganglion cells and inner retina (Hung et al., 2016), albeit at much lower efficiencies than by subretinal injections.”

• cites Hung et al., 2016 sourceNot checked — no full text of the source