Spatial and longitudinal tracking of enhancer-AAV vectors that target transgene expression to injured mouse myocardium
- Department of Cell Biology, Duke Regeneration Center, Duke University School of Medicine, United States
- Department of Surgery, Duke University School of Medicine, United States
- Department of Biomedical Engineering, Duke University, United States
- Department of Pharmacology, Vanderbilt University, United States
- Morgridge Institute for Research, United States
- Department of Cell and Regenerative Biology, University of Wisconsin-Madison, United States
Figures
Figure 1 with 1 supplement
Longitudinal tracking of tissue regeneration enhancer element (TREE)-directed transgene expression in injured mice by in vivo bioluminescence imaging (IVIS).
(A) Schematic illustration of study design. Albino BALB/c mice were systemically injected with AAV9 vectors packaging fLuc reporter cassettes directed by TREEs and a permissive promoter. Mice underwent ischemia/reperfusion (I/R) surgery at D61 and were imaged by IVIS at indicated time points. (B) Representative IVIS images indicate changes of expression over time and space for each vector. Cardiac region of interest (ROI) indicated by red box. n=2 mice. (C) Average radiance measured from cardiac ROIs plotted over days post-injury (dpi). Average radiance normalized to their baseline pre-injury was also plotted (right). n=2 mice. (D) Average cardiac radiance showed a transient increase in expression for both REN (left) and 2ankrd1aEN (right) after I/R injury, whereas sham-operated animals showed relatively constant expression. n=2 mice for I/R, n=3 mice for sham.
Figure 1—figure supplement 1
In vivo bioluminescence imaging (IVIS) imaging for tracking spatiotemporal expression of rAAV vectors.
(A) Schematic showing comparison of AAV9 vectors packaging either the strong, constitutively active chicken beta actin (CBA) promoter or minimal Heat shock protein 1a (Hsp1a) promoter to direct fLuc expression. n=5 mice/AAV group. (B) Representative IVIS images of mice injected with AAV containing either CBA (top) or Hsp1a (bottom) promoters. Red boxes indicate regions of interest (ROIs) marking cardiac, liver, and whole-body expression. (C) Average radiance measured from cardiac ROIs from CBA (top) or Hsp1a (bottom) promoters shows relatively consistent expression from 30 to 68 days post-AAV injection. Square, male mice. Circle, female mice. (D, E) Average radiance measured from liver (D) and whole-body (E) ROIs showed relatively consistent levels of expression over time for both promoters.
Figure 2 with 1 supplement
Liver-de-targeted AAV.cc84 capsid limits hepatic expression from tissue regeneration enhancer elements (TREEs).
(A) Schematic of experimental timeline, comparing AAV9 and AAV.cc84 capsids for systemic delivery of REN-Hsp1a::fLuc. Mice were in vivo bioluminescence imaging (IVIS) imaged in the weeks following AAV delivery and post-sham surgery. (B) Representative IVIS images of mice injected with either AAV9 (left) or AAV.cc84 (right) at 14 (top) and 21 days (bottom) post-AAV injection. Mice were also subdivided by biological sex to account for sex differences in AAV liver tropism. (C) Average radiance from liver regions of interest (ROIs) showed significantly higher expression in AAV9-transduced mice compared to AAV.cc84 through all timepoints (n=6 mice, Holm–Sidak multiple comparisons test). (D) Representative IVIS images of harvested organs at 42 days post-AAV demonstrate liver expression with AAV9 (left) while undetected with AAV.cc84 (right). (E) Vector genome quantification from collected liver samples reveals higher liver transduction with AAV9 compared to AAV.cc84 for both female and male mice.
Figure 2—figure supplement 1
AAV.cc84 capsid retains cardiac tropism while minimizing liver transduction.
(A) Representative in vivo bioluminescence imaging (IVIS) images of mice injected with either AAV9 (left) or AAV.cc84 (right) at 7 (top) and 35 days (bottom) post-AAV injection. (B) Average radiance in the heart was similar between AAV9 and AAV.cc84 (n=6 mice, Holm–Sidak multiple comparisons test). (C) Vector genome quantification from cardiac tissues showed similar levels of vector genomes between AAV9 and AAV.cc84 for both sexes (n=3 mice, Holm–Sidak multiple comparisons test). (D) Representative IVIS images of mice with regions of interest (ROIs) used to measure expression in head/neck and lower abdomen (white dashed box). (E) Average radiance measured in the head/neck (left) and lower abdomen (right) was similar between AAV9 and AAV.cc84 over the course of the study (n=6 mice, Holm–Sidak multiple comparisons test).
Figure 3 with 1 supplement
Post-injury delivery of AAV.cc84-packaged 2ankrd1aEN targets expression to cardiac injuries.
(A) Schematic of experimental timeline comparing expression between Hsp1a and 2ankrd1aEN when delivered at 4 dpi. (B) Representative in vivo bioluminescence imaging (IVIS) images of mice injected with Hsp1a (top) or 2ankrd1aEN- Hsp1a (bottom) after ischemia/reperfusion (I/R) injury. (C) Cardiac average radiance normalized to the 7 dpi time point increased over time with 2ankrd1aEN while remaining stable with Hsp1a (n=4 mice, Holm–Sidak multiple comparisons test). (D) Average cardiac radiance directed by 2ankrd1aEN was significantly higher in mice with I/R injury compared to sham at 7 dpi (n=4 mice, Welch’s t-test). (E) Average cardiac radiance was more significantly elevated in the first 21 days post-injury in mice with I/R injury compared to sham (n=4 mice, Mann–Whitney tests).
Figure 3—figure supplement 1
Delivery of AAV.cc84 packaged with 2ankrd1aEN after myocardial injury.
(A) Ischemia/reperfusion (I/R) surgery injury extent was assessed by ejection fraction via echocardiography to estimate injury prior to AAV delivery (n=4 mice). (B) Cardiac average radiance plotted for each individual mouse with I/R injury plotted over time with either Hsp1a::fLuc (gray) or 2ankrd1aEN- Hsp1a::fLuc (black). (C) Representative in vivo bioluminescence imaging (IVIS) images of sham-operated mice injected with AAV.cc84 packaged with 2ankrd1aEN- Hsp1a::fLuc.
Figure 4 with 1 supplement
Screening of AAV libraries for enriched capsids in injured myocardium.
(A) Schematic of AAV capsid library screening delivered systemically to mice with either sham (n=1 mouse) or ischemia/reperfusion (I/R) (n=2 mice) injury at 9 dpi. Two days later, hearts were biopsied to collect AAV genomes in either injured or remote tissues. (B) Capsid sequenced reads enriched in the injured tissues were plotted against sham animals. Each point represents a unique capsid. Wild-type AAV9 capsid is marked by blue arrow. (C) Representative fluorescence imaging of AAV9 (top) or variant capsid IR41 (bottom) delivering a self-complementary CBA::EGFP cassette at 16 dpi. Asterisks indicate infarct site, imaged at higher magnification in middle and right panel. Dashed white lines indicate the border zone region. Left scale bar, 1 mm. Middle and right scale bar, 100 um.
Figure 4—figure supplement 1
Screening AAV capsid libraries enriched in injured myocardium.
(A) Injury site-enriched capsids in I/R-injured mice. Each point represents a unique capsid. Top-left quadrant, capsids co-enriched at injury site. Wild-type AAV9 capsid is marked by an orange arrow. (B) Representative fluorescence imaging of variant capsids, IR42 (left) and IR43 (right), delivering a self-complementary CBA::EGFP cassette at 16 dpi. Asterisks indicate infarction area. Scale bar, 1 mm. (C) Immunofluorescence of infarct sites of mice transduced with either AAV9 (left) or IR41 (right) shows no colocalization of EGFP and Vimentin. Scale bar, 100 um.
Figure 5 with 1 supplement
Post-injury delivery of AAV.IR41 variant capsid enhances 2ankrd1aEN-directed expression in injured myocardium.
(A) Representative in vivo bioluminescence imaging (IVIS) images of mice with sham (top) or (bottom) surgery transduced with either AAV9 (left) or IR41 (right) packaged with 2ankrd1aEN- Hsp1a::fLuc (n=3–4 mice). (B, C) Cardiac average (B) and maximum (C) radiance was elevated in MI mice transduced with IR41 compared to AAV9 (n=3–4 mice, Holm–Sidak multiple comparisons test). (D) Viral genome quantification from heart tissues was elevated in MI mice injected with IR41 compared to AAV9 (n=3–4 mice, Holm–Sidak multiple comparisons test).
Figure 5—figure supplement 1
Post-injury delivery of variantAAV.IR41 variant capsid enhances 2ankrd1aEN-directed expression in injured myocardium over AAV9.
(A) Compiled in vivo bioluminescence imaging (IVIS) images of mice that underwent sham or MI surgery with AAV9 or IR41 transduced at 3 dpi. Mice were imaged at 7, 14, and 21 dpi. (B, C) Cardiac average (B) and maximum (C) radiance were statistically similar between AAV9 and IR41 in sham-operated mice.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Male and female C57BL/6J | The Jackson Laboratory | Strain 000664; RRID:IMSR_JAX:000664 | |
| Strain, strain background (M. musculus) | Male and female BALB/c | Charles River | Strain 028; RRID:IMSR_CRL:028 | |
| Recombinant DNA reagent | AAV9: CBA::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV9: Hspa1a::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV9: REN- Hspa1a::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV9: 2ankrd1aEN- Hspa1a::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV.cc84: REN- Hspa1a::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV.cc84: Hspa1a::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV.cc84: 2ankrd1aEN - Hspa1a::fLuc | This paper | Available from corresponding authors | |
| Recombinant DNA reagent | AAV.IR41: 2ankrd1aEN - Hspa1a::fLuc | This paper | IR41 VR4 peptide sequence: GPGVGAR | Available from corresponding authors |
| Recombinant DNA reagent | AAV.IR42: 2ankrd1aEN - Hspa1a::fLuc | This paper | IR42 VR4 peptide sequence: ASRNVVT | Available from corresponding authors |
| Recombinant DNA reagent | AAV.IR43: 2ankrd1aEN - Hspa1a::fLuc | This paper | IR43 VR4 peptide sequence: SDSQYVQ | Available from corresponding authors |
| Recombinant DNA reagent | AAV9 capsid library | Gonzalez et al., 2022; Gonzalez et al., 2023 | ||
| Cell line (human) | HEK293 | ATCC; UNC Viral Vector Core; Gonzalez et al., 2022; Gonzalez et al., 2023 | RRID:CVCL_0045 | Obtained from UNC Viral Vector Core for production of recombinant AAV at Duke, see Asokan lab citations |
| Antibody | Rabbit anti- GFP | Invitrogen | #A-11122; RRID:AB_221569 | 1:1000 |
| Antibody | Mouse anti-vimentin | DSHB | #40E-C; RRID:AB_528504 | 1:100 |
| Antibody | Goat anti-rabbit IgG Alexa Fluor 488 | Invitrogen | #A-11008; RRID:AB_143165 | 1:500 |
| Antibody | Goat anti- mouse IgG Alexa Fluor 555 | Invitrogen | #A21422; RRID:AB_2535844 | 1:500 |
| Sequence-based reagent | ITR F | This paper | PCR primers | 5’ AACATG CTACGC AGAGAG GGAGTGG 3’ |
| Sequence-based reagent | ITR R | This paper | PCR primers | 5’CATGAGA CAAGGA ACCCCT AGTGAT GGAG 3’ |
| Sequence-based reagent | AAV9 lib amp F | This paper | PCR primers | 5’ AGCACG GTCCAGGT CTTCAC 3’ |
| Sequence-based reagent | AAV9 lib amp R | This paper | PCR primers | 5’ ATGTCAG TCTAGAC CAAAGTT CAACTGA AACGAAT TAAACGG 3’ |
| Sequence-based reagent | WPRE-bGH F | This paper | PCR primers | 5’ CTTCGCC CTCAGAC GAGTCGGA 3’ |
| Sequence-based reagent | WPRE-bGH R | This paper | PCR primers | 5’ TGGCTGG CAACTAG AAGGCACA 3’ |
| Commercial assay or kit | PureLink Genomic DNA mini kit | Invitrogen | K182002 | |
| Chemical compound, drug | D-luciferin, potassium salt | Gold Biotechnology | LUCK-1G |
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Spatial and longitudinal tracking of enhancer-AAV vectors that target transgene expression to injured mouse myocardium
eLife 14:RP107148.
https://doi.org/10.7554/eLife.107148.3
