A dual-reference study design for understanding and improving AAV genome size analysis
© 2024 The Authors. Electrophoresis published by Wiley-VCH GmbH..
Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2024 |
|---|---|
| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2024 |
|---|---|
| Enthalten in: |
Electrophoresis - (2024) vom: 15. März |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Sun, Yali [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
AAV genome integrity |
|---|
| Anmerkungen: |
Date Revised 15.03.2024 published: Print-Electronic Citation Status Publisher |
|---|
| doi: |
10.1002/elps.202400011 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM369783891 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM369783891 | ||
| 003 | DE-627 | ||
| 005 | 20240315234850.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 240315s2024 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1002/elps.202400011 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1331.xml |
| 035 | |a (DE-627)NLM369783891 | ||
| 035 | |a (NLM)38488707 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Sun, Yali |e verfasserin |4 aut | |
| 245 | 1 | 2 | |a A dual-reference study design for understanding and improving AAV genome size analysis |
| 264 | 1 | |c 2024 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Revised 15.03.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status Publisher | ||
| 520 | |a © 2024 The Authors. Electrophoresis published by Wiley-VCH GmbH. | ||
| 520 | |a Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a AAV genome integrity | |
| 650 | 4 | |a dual reference | |
| 650 | 4 | |a microfluidic capillary electrophoresis | |
| 650 | 4 | |a ssAAV | |
| 650 | 4 | |a ssDNA 7K Ladder | |
| 700 | 1 | |a Lu, Zhi-Xiang |e verfasserin |4 aut | |
| 700 | 1 | |a Miller, Michael |e verfasserin |4 aut | |
| 700 | 1 | |a Valcour, Ying |e verfasserin |4 aut | |
| 700 | 1 | |a Khimani, Anis H |e verfasserin |4 aut | |
| 700 | 1 | |a Bauer, Jana |e verfasserin |4 aut | |
| 700 | 1 | |a Salomon, Michael |e verfasserin |4 aut | |
| 700 | 1 | |a Tong, Yanhong |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Electrophoresis |d 1988 |g (2024) vom: 15. März |w (DE-627)NLM012613363 |x 1522-2683 |7 nnns |
| 773 | 1 | 8 | |g year:2024 |g day:15 |g month:03 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1002/elps.202400011 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |j 2024 |b 15 |c 03 | ||