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. 2010 May 20;3(3):81–89. doi: 10.1111/j.1752-8062.2010.00190.x

Comparative Cardiac Gene Delivery of Adeno‐Associated Virus Serotypes 1–9 reveals that AAV6 Mediates the Most Efficient Transduction in Mouse Heart

Carmela Zincarelli 1, Stephen Soltys 1,2, Giuseppe Rengo 1,2,3, Walter J Koch 1,2, Joseph E Rabinowitz 1
PMCID: PMC3962265  NIHMSID: NIHMS196377  PMID: 20590676

Abstract

Cardiac gene transfer is an attractive tool for developing novel heart disease treatments. Adeno‐associated viral (AAV) vectors are widely used to mediate transgene expression in animal models and are being evaluated for human gene therapy. However, it is not clear which serotype displays the best cardiac tropism. Therefore, we curried out this study to directly compare AAV serotypes 1–9 heart transduction efficiency after indirect intracoronary injection. AAV‐cytomegalovirus immediate early enhancer promoter (CMV)‐luciferase serotypes 1–9 were injected in the left ventricular cavity of adult mice, after cross‐clamping the ascending aorta and pulmonary artery. An imaging system was used to visualize luciferase expression at 3, 7, 21, 70, and 140 days postinjection. Echocardiography was performed to evaluate cardiac function on day 140. At the end of the study, luciferase enzyme activity and genome copies of the different AAV serotypes were assessed in several tissues and potential AAV immunogenicity was evaluated on heart sections by staining for macrophage and lymphocyte antigens. Among AAV serotypes 1–9, AAV6 showed the best capability of achieving high transduction levels in the myocardium in a tissue‐specific manner, whereas the other serotypes had less cardiac transduction and more extracardiac expression, especially in the liver. Importantly, none of the serotypes tested with this marker gene affected cardiac function nor was associated with inflammation.

Keywords: adeno‐associated virus, gene therapy, AAV cardiac tropism, indirect intracoronary gene delivery

Introduction

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide. 1 , 2 Chronic heart failure, which is the end result of several cardiac diseases, represents an important clinical challenge because of its rising mortality and incidence. 2 , 3 Despite considerable improvement in the pharmacological treatment of the cardiovascular diseases, such as heart failure, the above challenges make it clear that innovative strategies are warranted. Accordingly, gene therapy has been considered an alternative and novel experimental therapeutic approach for heart disease. In fact, this strategy is able to modify gene‐expression profles or correct specifc gene defects that lead to several cardiomyopathies. Different methods, such as naked DNA, 4 , 5 liposomes, 6 , 7 and viral vectors, 8 , 9 , 10 , 11 have been used for this purpose. Among the viral vectors, adeno‐associated virus (AAV) serotypes have been identified as promising tools for cardiac gene delivery since they own several advantages over other vectors: they are not pathogenic in humans 12 and they provide significantly longer transgene expression 10 including a sustained myocardial transduction. 9 , 10 , 11 , 13

All AAVs are Dependoviruses (belonging to the Parvoviridae viral family). They have a small nonenveloped protein capsid 14 containing a single‐stranded DNA genome encoding two genes: the rep gene that encodes four proteins involved in DNA replication, and the cap gene that encodes three proteins which, via alternative splicing, make up the protein coat of the virus. 15 New AAV serotypes are continuously being identified. At present, over 100 AAV variants have been isolated. 16 , 17 , 18 , 19 , 20 Until recently, the majority of studies used AAV2‐based vectors for targeting cardiac muscle. However, AAV2 vector‐mediated gene transfer is limited by the presence of neutralizing antibodies (NAbs) in humans due to prior exposure to wild‐type virus. 21 Recently, comparative cardiac tropism evaluations of AAV serotypes have been examined in several studies: Wang et al. 22 compared the efficiency of AAV1, 2, 5, 6, 7, and 8 after a single injection via intraperitoneal (IP) or intravenous routes in neonatal and adult mice and hamsters. They showed, after 1 month from tail vein injection of AAV8‐green fluorescent protein (GFP), strong GFP expression in the heart and, to a lesser degree, in several skeletal muscles, while intravenous injection of other AAV serotype vectors including AAV1, 2, 5, and 6 resulted in much lower GFP expression in muscle and heart. In addition, they showed a strong cardiac gene expression given by AAV1 and AAV6 2 months after gene delivery by IP injection in neonatal mice. 22 Su et al. 23 injected into mouse myocardium AAV serotypes 1–5 showing that AVV vectors serotype 1 mediates earlier and higher gene expression in hearts than other serotypes. 23 Inagaki et al. 24 compared AAV9 and AAV8 cardiomyocyte transduction following tail vein injection in mouse. In this study, the authors claimed that AAV9 vectors share the robustness of AAV8; both vectors show very high liver transduction efficiency, irrespective of whether vectors are administered intravascularly or extravascularly and substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, AAV9 transduced myocardium 5‐ to 10‐fold higher than AAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 × 1011 particles per mouse. 24 Pacak et al. 25 compared rAAV2/1 with rAAV2/8 and rAAV2/9 vectors in their respective abilities to transcend vasculature and transduce myocardium following intravenous delivery of 1 × 1011 vector genomes in neonatal mice. They found that both rAAV2/8 and rAAV2/9 are able to transduce myocardium at 20‐ and 200‐fold (respectively) higher levels than rAAV2/1. 25

Palomeque et al. 26 investigated the time course of expression of AAV serotypes 1–8 in rat heart after direct intramyocardial injection. The authors showed that AAV1, 6, and 8 had the highest efficiency in transducing rat hearts, although AAV1 and AAV6 vectors induced rapid and robust expression reaching a plateau at 4 weeks while AAV8 continued increasing until the end of the study. AAV2, 5, and 7 vectors were slower to induce expression of the reporter gene, but did reach levels of expression comparable to AAV1 and AAV6 vectors after 3 months. 26

From the above studies, it becomes evident that identifcation of the serotype with the highest tropism for the heart is hampered by the fact that different routes of injection, different animals’ age, as well as virus titers, and serotype numbers have been used. The present study is the first, to our knowledge, to test all the characterized AAV serotypes 1–9 packaged and injected into heart in the same way (indirect intracoronary delivery), in order to investigate diferences in cardiac tropism, kinetics of expression, inflammatory responses, and effects on cardiac function. Moreover, using luciferase as a transgene, the activity of transgenic protein was evaluated following IP injection of its substrate (luciferin), and the measurements were performed sequentially in the same animal. 27 , 28 , 29 This allowed assessment of individual variations in expression of the same serotype in the same animal over time. We have found that AAV6 is capable of achieving the highest transduction levels specifically in the myocardium between the nine serotypes tested after indirect intracoronary gene delivery, without negatively affecting cardiac function or causing local infammation.

Materials and Methods

Cell lines, plasmids and production of recombinant virus

Recombinant AAV vectors were generated, purif ed, and titered as previously described. 30 Vector containing AAV2‐inverted terminal repeats (ITRs) and the CMV promoter driving luciferase transgene was packaged within AAV serotypes 1–9 ( Figure 1A ). Total vector genome number have been reported elsewhere, 30 except for AAV1 Luc 8.2 × 1011/mL, AAV4 Luc 7.0 × 1012/mL, AAV5 Luc 3.5 × 1011/mL, and AAV8 Luc 8.2 × 1011/mL.

Figure 1.

Figure 1

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Study design. (A) Diagram of packaging vector containing AAV2‐ITRs and the CMV driven luciferase transgene. This transgene was packaged within AAV serotypes 1–9. (B) After indirect intracoronary injection mice were imaged according to the above schedule and sacrificed after 140 days.

Indirect intracoronary gene delivery

All animal experiments were conducted in accordance with the IACUC of T omas Jef erson University. Ten‐week‐old male Balb/C mice were purchased from Charles River Breeding Laboratories (Wilmington, MA, USA). Mice were anesthetized with isofluorane induction and endotracheally intubated. Animals were placed supine and ventilated with isof uorane (1.5%) vaporized in 100% O2 using volume controlled ventilator (Harvard Apparatus, Holliston, MA, USA). Core temperature was monitored throughout the procedure using a thermocouple thermometer (Type T, Digi‐Sense, Oakton Instruments, Vernon Hills, IL, USA). The chest was opened with a midline thoracotomy till the fourth–fifth rib, while mice were cooled with a water pad to a core temperature of 25°C and then a vascular clamp was used to occlude both proximal aorta and pulmonary artery. A 31‐gauge needle was advanced into the lef ventricular (LV) cavity to deliver 1.3 × 1010 virus particles (VPs) of AAV‐CMV‐luciferase serotype 1–9 (n= 5 for each serotype) with 2 μL of substance P (0.2 μg‐Sigma Chemical Co, St Louis, MO, USA) in a total volume of 100 μL of saline while control animals (n= 5) were injected with 100 μL of saline with or without pharmacological agents. The virus was delivered as a bolus immediately after cross‐clamping and the clamp was maintained for 45 seconds after vector injection. After releasing the vascular clamp, the mice were rewarmed to 37°C using a heating pad, the chest and skin were closed with 6–0 silk suture; mice were extubated and recovered in their cages.

In vivo animal imaging

Imaging was performed as previously described. 30 For each image, the visual output represents the “radiance” and the units are photons/sec/cm2/sr. This refers to the number of photons released per second from each square centimeter of tissue and radiating into a solid angle of one steradian (SR). All animals were imaged on a schedule of 3, 7, 21, 70, and 140 days after AAV indirect intracoronary injection ( Figure 1B ). Bioluminescent pictures are pseudocolor images referred to as the luminescent image overlaid on a photographic one. On the right side of the image is a color bar that shows the relationship between the pseudocolors in the image and the numerical values of photons released. It is a reverse rainbow color table where violet is assigned to the lowest number in the array, red to the highest, and all the spectral colors of the rainbow to values in between.

In Vivo quantifying luciferase expression

At each time point a region of interest was used to surround the heart, and at 140 days postinjection also the liver area, of each animal in order to quantify the total flux (TF) expressed as photons/sec/cm2/sr being released by luciferase activity from these zones. For each AAV serotype, the mean value was determined averaging the TF emitted at each time point from the mice injected with that serotype.

Echocardiography

At 140 days postindirect intracoronary injection, echocardiographic studies were performed for each animal using an ultrasonographic system (VEVO‐770; VisualSonic, Toronto, Canada, UK) basally and after isoproterenol stimulation (10 μg/kg/body weight). After anesthesia with Avertin (8 μL/g body weight, Sigma‐Aldrich, St Louis, MO, USA) via intraperitoneal injection, mice were placed in a supine position. A 12‐MHz transducer was applied to the left hemithorax. Two‐dimensional targeted M‐mode imaging was obtained from the short‐axis view. M‐mode measurements of LV end‐diastolic and end‐systolic diameter and LV anterior and posterior wall thickness were made using the leading‐edge convention of the American Society of Echocardiography. The percentage of left ventricular fractional shortening (LVFS) was calculated as LVFS (%) = (LVEDD − LVESD)/LVEDD × 100, where LVEDD and LVESD indicate LV end‐diastolic and end‐systolic diameter, respectively.

Luciferase enzyme activity and vector genome copies

At 140 days after indirect intracoronary injection of AAV‐CMV‐luciferase 1–9 serotypes, animals were sacrificed and selected organs dissected: brain, heart, lung, liver, kidney, testes, and quadriceps. Luciferase enzyme activity and viral genome copy number were evaluated from those tissues as previously described. 30

Immunostaining

At 140 days after indirect intracoronary gene delivery, the spleen and the heart from mice injected with saline or AAV1, 2, 6, 7, 8, and 9 serotypes were harvested. The spleens and hearts (cut into three short‐axis slices) were immediately fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4), embedded in paraffin and sectioned (5 μm). Sections were deparaffinize in xylene, rehydrated in a graded series of alcohols, and incubated in 3% H2O2 (in PBS) to quench endogenous peroxidase activity. After being rinsed in PBS, the sections were incubated with block buffer and 0.1% Triton. Lymphocytes’ staining was performed using Alexa fluor 488 antimouse CD3 (Biolegend, San Diego, CA, USA), while for macrophages’ detection a rat antimouse F4/80 (clone A3–1; Abcam, Cambridge, MA, USA) antibody was used. Biotinylated goat antirat IgG (Vector Laboratories, Burlingame, CA, USA) was then applied for 1 hour at room temperature. After incubation with avidin‐biotin complex (Vector Laboratories, Burlingame, CA, USA), immunoreactivity was visualized by incubating the sections with 3,3‐diaminobenzidine tetrahydrochloride (DAKO, Carpinteria, CA, USA) to produce a brown precipitate.

Statistical analysis

Data are summarized as mean ± SEM. Comparisons were made using t‐test or ANOVA as appropriate. A Bonferroni correction was applied to the probability values whenever multiple comparisons arose. Statistical analysis was performed with the Graph PadPrism software. For all tests, p < 0.05 is considered statistically significant after Bonferroni corrections if needed.

Results

Onset and localization of transgene expression by in vivo imaging

We have directly compared AAV serotypes 1–9 in their ability to transduce myocardium in vivo after indirect intracoronary gene delivery, 31 using substance P as permeabilizing agent. 32 We tested all AAV serotypes carrying CMV‐luciferase by imaging animals at 3, 7, 21, 70, and 140 days postgene delivery ( Figure 1B ). Direct examination of those images revealed large differences in thoracic bioluminescence among serotypes; therefore we segregated animals injected with different AAV serotypes into three groups using different intensity scales ( Figure 2 ). Three days after AAV injection, expression was observed in mice injected with AAV serotypes 6, 7, and 9 ( Figure 2A and B ). More specifically, AAV7‐ and AAV9‐injected animals displayed upper abdominal luciferase bioluminescence ( Figure 2A and B ), while AAV6‐injected mice showed higher luciferase expression localized to the thoracic cavity ( Figure 2A ). AAV serotypes 1, 2, 4, and 8 did not show any detectable expression until 7 days postinjection ( Figure 2B ). AAV3 and AAV5 expression was lower than other groups; only at 21 days postinjection they showed bioluminescence above background in the thoracic cavity ( Figure 2C ).

Figure 2.

Figure 2

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In vivo imaging of luciferase after indirect intracoronary injection of AAV serotypes 1–9. (A) High‐expression group: for AAV serotypes 6 and 9 the expression range was 4,000–200,000 photons/sec/cm2/sr. (B) Medium expression group includes AAV1, 2, 4, 7, and 8. For this group, the range of expression was 4,000–35,000 photons/sec/cm2/sr. (C) Low‐expression group: for AAV3 and AAV5 the expression range was 1,800–15,000 photons/sec/cm2/sr. For each serotype injected, we show two representative mice. Images are shown at 3, 7, 21, 70, and 140 days post virus injection, as indicated by numbers at left of each panel.

In the medium expression group, AAV1 and AAV4 had similar localization patterns on the ventral axis; these animals emitted light from the thoracic cavity without light emission from the upper abdomen ( Figure 2B ). AAV serotypes 2, 7, and 8 had overlapping patterns: these serotypes emitted light from the thorax and upper abdomen on the ventral axis and this pattern of expression remained throughout the study (140 days postinjection) ( Figure 2B ). A comparison of thoracic and abdominal expression demonstrated AAV2 had higher luciferase expression in the thorax, while AAV7 and AAV8 owned higher levels in the upper abdomen. The high‐expression group consisted of AAV6 and AAV9; AAV6‐injected animals showed the highest level of bioluminescence in the thorax without significant abdominal expression within the indicated light range ( Figure 2A ). In contrast, AAV9‐injected animals displayed significant bioluminescence in the upper abdominal cavity, as well as expression in the thorax.

In vivo levels and kinetics of luciferase expression

In order to quantify the light released, representing luciferase activity, from the heart of each animal, we calculated the number of TF in photons/sec/cm2/sr emitted from the thorax at each time point, after gene delivery, using Xenogen IVIS100 (Caliper Life Sciences, Hopkinton, MA) imaging software ( Figure 3A ). In the low‐expression group, AAV3 and AAV5 showed their highest level at 7 and 21 days after injection. Within the medium expression group, AAV1‐injected animals showed a mean peak of expression at 70 days postinjection (2.1 × 104± 5.2 × 103 TF), with a 23% decrease by 140 days (1.7 × 104± 1.7 × 103 TF), while AAV2‐injected animals showed a time‐dependent expression increasing up to 140 days postinjection (4.0 × 104± 1.4 × 104 TF) ( Figure 3A ); AAV4 heart expression had a slow increase up to 70 days postinjection (1.3 × 104± 5.4 × 103 TF) followed by a 38% decrease at 140 days (8.02 × 103± 2.1 × 103 TF). AAV7‐injected animals also demonstrated their peak at 70 days postinjection reaching 3.8 × 104± 9.5 × 103 TF and then declining to 140 days (1.8 × 104± 2.5 × 103 TF). In contrast, AAV8 heart expression increased to the last time point examined (3.9 × 104± 9.6 × 103 TF) ( Figure 3A ). In the high‐expression group, AAV9‐injected animals had a rapid increase of heart expression between 3 and 21 days postinjection, after which bioluminescence slowly increased reaching its maximum level at 140 days after vector delivery (7.2 × 104± 3.6 × 104 TF) ( Figure 3A ). Finally, for AAV6‐injected mice, heart expression peaked at 21 days reaching the highest level (7.9 × 105± 3.5 × 105 TF) followed by a slow decline of 29% by 140 days postinjection (5.6 × 105± 1.3 × 105 TF) ( Figure 3A ).

Figure 3.

Figure 3

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Kinetic of luciferase transgene. (A) Total flux (photons/sec/cm2/sr) released by luciferase bioluminescence emitted from an area surrounding the heart at different time point from gene delivery. For each serotype, the mean flux was determined for each animal at each time point. (B) Photons/sec/cm2/sr released from the heart and liver area by the luciferase bioluminescence at 140 days after cardiac gene delivery of AAV1–9 serotypes. The values used are from the ventral images and are shown as mean ± SEM (n= 5 for each group at each time point); *p < 0.05 versus all heart values, ANOVA analysis and Bonferroni test were used among all groups.

In addition, we compared the number of TF emitted from the thorax with the upper abdominal area TF for each animal at 140 days after indirect intracoronary injection ( Figure 3B ).

Remarkably, AAV6 not only showed the highest heart expression, but also a low amount of bioluminescence emitted from the liver area, with a thoracic to upper abdominal TF ratio of 170.4 ( Figure 3B ). AAV8 and AAV9 showed a lower ratio (1.5) since their bioluminescence was less in the thorax and more in the upper abdomen compared to AAV6. For AAV1 and AAV4 serotypes the ratio was 11.3 and 7.5, while for AAV2 it was as low as 2.38. Only AAV serotype 7 emitted more TF from the liver area compared to thorax, a ratio of 0.43 ( Figure 3B ).

Luciferase enzyme activity in several tissues

To further analyze the transduction efficiency of different AAV serotypes, several organs from injected mice were harvested and proteins examined for luciferase enzyme activity at 140 days postinjection ( Figure 4 ). The amount of RLU/mg of total protein emitted from those tissues of AAV serotype‐injected animals conf rmed the patterns of localization observed by imaging. The indirect intracoronary delivery resulted in all serotypes having the greatest number of RLU/mg of total protein in the heart. Secondary expression was seen in the liver mainly for AAV2, 7, 8, and 9 or in the lung for AAV4 and AAV6 ( Figure 4 ). The ratios of luciferase enzyme activity in heart and liver are similar to the ratio of TF emitted from the thoracic and upper abdominal cavities. Interestingly, AAV7‐mediated luciferase expression in the heart was only 1.7‐fold greater than the liver; this was followed by AAV8 with an 11.7‐fold difference and AAV9 with an 18‐fold dif erence in luciferase enzyme activity between heart and liver. In contrast, AAV6 showed the absolute highest level of luciferase enzyme activity in the heart with 500‐ and 4000fold greater expression when compared to lung and liver, respectively. In general, we observed a direct relationship between luciferase protein expression (RLU/mg total protein) in various tissues and the patterns of light production observed with imaging.

Figure 4.

Figure 4

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Biodistribution of luciferase enzyme activity. Luciferase activity (in relative light units [RLU] per mg of total protein) was determined in selected dissected tissue 140 days after indirect intracoronary injection of 1.3 × 1010 particles of AAV1–9 CMV‐luciferase constructs into adult mice. Data are presented as mean ± SEM (n= 5 for each group). #p < 0.05 versus all heart values; *p < 0.001 versus AAV1, 2, 3, 4, 5, 6, and 8 liver values; &p < 0.05 versus AAV1, 2, 3, 4, 5, 6, and 8 liver values; ANOVA analysis and Bonferroni test were used among all groups.

Viral genome copy number remaining in selected tissues

We next examined the viral genome copy number in several tissues against total genomic DNA in those tissues ( Figure 5 ). All nine AAV serotypes showed the highest luciferase genome copy number in heart tissue: AAV6 showed the highest value (3.1 × 106± 1.4 × 106 viral genomes/μg of total DNA) followed by AAV2 and AAV9 (6.0 × 105± 5.0 × 105 and 3.3 × 105± 1.3 × 105 viral genome copies/μg of total DNA, respectively) ( Figure 5 ). In the medium expression group, after AAV2, the hierarchy of vector genome copy number was in the following order: AAV serotypes 1, 8, 7, and 4. Interestingly, AAV7 vector genome copy number in the heart was comparable to that observed in the liver (8.5 × 104± 7.0 × 104 and 7.3 × 104± 9.0 × 103, respectively) with a heart/liver ratio of 1.1 ( Figure 5 ), while AAV9 genome copy number was 5‐fold higher in the heart than in the liver. In contrast, for AAV6 the ratio heart/liver was of 516:1. Finally, AAV3 and AAV5 serotypes showed the lowest vector genome copies in the heart ( Figure 5 ).

Figure 5.

Figure 5

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Vector genome copy numbers in selected tissues. Persistence of viral genomes in selected dissected tissues 140 days after indirect intracoronary injection of 1.3 × 1010 particles of AAV serotypes 1–9. Values are shown as mean ± SEM (n= 5 for each group). *p < 0.001 versus all heart values; &p < 0.001 versus AAV1, 3, 4, 5, 6; #p < 0.05 versus AAV2 and AAV8. ANOVA analysis and Bonferroni test were used among all groups.

Effect of viral transduction on heart function by echocardiography

Since there are potential adverse effects from viral exposure, basal, and stress transthoracic echocardiography were performed to evaluate myocardial function at 140 days after indirect intracoronary injection. As shown in Figure 6 , none of the AAV serotypes tested had any detectable ef ect on cardiac contractility at 140 days after indirect intracoronary injection, neither basally nor after isoproterenol stimulation. LV fractional shortening (%FS) was found to be equivalent between AAV‐injected and control (saline) groups ( Figure 6A ). Moreover, LV end‐diastolic diameter did not significantly differ among AAV‐ and saline‐injected mice ( Figure 6B ). Representative M‐mode echocardiographic recordings from control and AAV6‐injected animals at 140 days are shown in Figure 6C .

Figure 6.

Figure 6

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AAV cardiac gene delivery does not negatively affect heart function. (A) Left ventricular (LV) fractional shortening (%FS) 140 days after indirect intracoronary injection of AAV1–9 or saline, as control (C). (B) LV chamber dimensions were similar among mice injected with saline and animals injected with AAV1–9. (C) Representative raw traces of M‐mode echocardiography 140 days after AAV6‐CMV‐Luciferase indirect intracoronary gene delivery or saline, basal and after isoproterenol stimulation. Bar = 5 mm. Values are shown as mean ± SEM (n= 5 for each group).

Immunological cell infiltration in the heart after AAV gene delivery

Immunofluorescence was performed in mice with the highest heart transgene expression (AAV1, 2, 6–9) to detect CD3+ cells in heart sections 140 days postgene delivery. CD3 was used as a general marker of T lymphocytes, since nearly all CD4+ and CD8+ T cells express CD3. 33 No inflammatory infiltrates were observed in the heart 140 days postinjection for any serotype ( Figure 7A ). In addition, F4/80 antigen was used as a marker for macrophage infiltration. Immunohistochemistry performed on myocardium of AAV serotypes 1, 2, 6–9 injected hearts indicated an absence of significant infiltration similar to controls ( Figure 7B ). Taking together these results indicate that AAVs are not inducing a significant immunoreaction in the myocardium, at least at the dose and time point examined in the present study.

Figure 7.

Figure 7

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Immunological reaction after AAV‐mediated gene delivery. (A) Immunofluorescence for CD3+ cells in heart sections of mice injected with AAV serotypes 1, 2, 6–9 after 140 days from gene delivery or with saline. (B) Immunostaining for F4/80 macrophage antigen on heart section of mice injected with AAV serotypes 1, 2, 6–9 or saline, after 140 days from gene delivery. The black arrows indicate macrophage infiltration clearly visible in the spleen section used as positive control. Bar = 100 μm. Magnifi cation, ×20.

Discussion

AAV vectors represent promising therapeutic tools for gene therapy of cardiovascular diseases: they target the underlying genetic modification and can support long‐term and stable gene expression with minimal immunogenic and inflammatory host responses, providing researchers and clinicians with a powerful group of gene delivery tools. 34 From the clinical and therapeutic points of view, knowledge of transduction efficiency and kinetics (onset and duration of expression) in the myocardium is essential. For this purpose, in the present study, we sought to investigate the heart tropism of nine AAV serotypes (1–9) following cardiac gene delivery in adult mice.

To compare AAV serotypes’ efficiency in cardiac transduction we used indirect intracoronary gene delivery, since this approach has been proved to obtain high yield and physiologically relevant gene transfer to the murine heart. 31 In particular, we chose this technique because (1) it is broadly used, as well as direct intracoronary injection, for cardiac gene delivery in animals and represents an applicable methodology for the human clinical setting, and (2) the main alternative method (direct intramyocardial injection) has the big limitation to produce transgene expression only in the injection site, potentially masking differences in tropism among the AAV serotypes. Moreover, we injected substance P, a permeabilizing agent, together with the vector, in order to increase transgene expression into the myocardium. 32

The overall techniques applied in the present study to measure the expression of each AAV serotype provide a comprehensive picture of the different properties owned by the nine AAVs tested. Since we designed each helper plasmid in the same genetic background, used the same methods of production, purification, and delivery for each vector, the differences in vector tropism, onset and efficiency of expression can be attributed exclusively to the virion shell.

With in vivo imaging, we pointed out the differences among serotypes in terms of onset of gene expression profile: at 3 days after gene delivery, cardiac luciferase bioluminescence was already detectable only for AAV6. Of note, also AAV9 and AAV7 bioluminescence was depicted as early as 3 days after transgene delivery, but was localized in the upper abdomen. Moreover, the in vivo imaging showed for AAV6 higher cardiac expression levels than all the other serotypes at all time points analyzed. The strong cardiac tropism owned by AAV6 was also confirmed by the highest levels of luciferase protein activity and genome copy number found in the heart tissue at 140 days postgene delivery. Remarkably, concerning the specificity of this expression, AAV6 is associated with lower liver expression compared to AAV2, 7, 8, and 9. Although serotypes such as AAV1, 3, 4, and 5 showed similar to lower liver transduction compared to AAV6, their cardiac expression was strongly reduced in comparison with AAV6. Taken together these results suggest that AAV6 is the serotype with the earliest onset of expression, the highest myocardial transduction, and the most cardioselective expression compared to all other serotypes.

Our finding is consistent with Gregorevic et al., 35 who showed that adult mice receiving rAAV6—CMV‐lacZ presented high cardiomyocytes transduction after intravenous injection. Moreover, a recent study performed by our group 30 demonstrated that after tail vein injection of AAV1–9, AAV6 had a bias toward the heart with higher expression in the heart compared to the liver. 30 In another elegant work, Palomeque et al. 26 compared the gene expression among AAV serotypes 1–8 after direct injection into rat myocardium, and consistent with our results, showed that at all time points studied, AAV6, together with AAV1 and AAV8 demonstrated the highest efficiency in transducing rat hearts in vivo.

After AAV6 the second serotype with high heart transduction was AAV9. In accordance with our results, Inagaki et al. 24 compared AAV9 and AAV8 cardiomyocyte transduction after tail vein injection in mouse and demonstrated that AAV9 had higher myocardium transduction than AAV8. In our previous study, 30 we showed that AAV9 and AAV6 were the serotypes with the highest heart transduction. However, AAV9 showed high liver expression both after indirect intracoronary delivery, as demonstrated in the present study, and after tail vein injection. 30

In vivo imaging, coupled with the luciferase protein assay and the genome copy number, revealed cardiac transduction, besides AAV6 and AAV9, for serotypes such as AAV1, 2, 4, 7, and 8; their level of expression in the heart was far lower compared to AAV6 and associated with a variable transduction level in the liver. Interestingly, AAV serotypes 2, 4, and 6 also showed a modest lung tropism albeit without any statistical significance.

AAV3 and AAV5 serotypes were associated with a very low transduction level in the heart: their bioluminescence level was comparable with that of saline‐injected mice, and this lack of expression was confirmed by low levels of luciferase enzyme activity and genome copies not only in the heart but also in other tissues examined. Neither Du et al. 36 nor Wang et al. 22 were able to detect any gene expression with AAV5 in the cardiac tissue. Furthermore, with regard to the low expression observed for the AAV3 serotype, our results are in agreement with those from Palomeque et al., 26 who showed that the gene expression mediated by AAV3 was poor during the entire 6‐month‐long study, at each time point analyzed. Accordingly, Du et al. 36 observed no significant gene expression with AAV3, neither in vitro in murine and human cardiomyocytes nor in vivo in mouse hearts.

After indirect intracoronary gene delivery, the in vivo imaging did not show any bioluminescence emitted elsewhere except thorax and upper abdomen for all serotypes tested. Indeed, the quantification of luciferase protein activity and viral genome copy number in tissues such as kidney, quadriceps, brain, and testes did not reveal a significant AAV localization in these tissues.

In addition, indirect intracoronary delivery of AAV vectors was not associated with increased inflammatory infiltrates on histological heart section compared with animals receiving saline. With the amount of vector particles used in the present study, neither lymphocytes infiltration nor macrophages recruitment were found in the heart tissue of animals injected. These findings are in line with previous studies 37 and are consistent with our echocardiographic results. In fact, both basal and isoproterenol‐stimulated %FS, as well as LV end‐diastolic diameter, were found to be comparable between AAV‐ and saline‐injected mice. Remarkably, AAV6 serotype, albeit its highest level of heart transduction ef cacy compared to all serotypes, did not have any negative effect on cardiac function. Of note, at the best of our knowledge, this is the first study to investigate the effects of all characterized AAV serotypes 1–9 on cardiac function, either basally or after isoproterenol stimulation.

In the present study, we have not investigated AAV serotypes‐mediated transgene expression in different cell types within the heart or other organs. However, in a recent study by our group 38 , we have shown that AAV6‐GFP cardiac gene delivery resulted in transgene expression both in the cardiomyocyte and noncardiomyocyte fractions of the heart. Transgene expression in cardiac cells other than myocytes warrants further investigation aiming to evaluate AAV serotypes’ tropism for different cell types within organs.

In conclusion, AAV6 appears to be the most capable serotype to achieve a global and robust cardiac transduction among all other serotypes tested; AAV6 expression is also highly cardioselective, since the tropism for cardiac tissue is accompanied by low transgene levels in other extracardiac tissues. Finally, we have shown that AAV6 serotype does not affect cardiac function. These results suggest that AAV6 might be the best choice for cardiac gene delivery, since its efficacy in cardiac transduction and safety make it suitable for clinically relevant gene transfer application for cardiovascular diseases.

Acknowledgments

This work was supported by grants from the American Heart Association AHA F69103 (JER), National Institutes of Health (NIH) grant RO1 HL091096 (JER) and HL56205 (WJK), and by funding from the Pennsylvania Department of Health, Pennsylvania Tobacco Grant A75301 (WJK). GR was supported by fellowship from the American Heart Association (Great Rivers Affiliate).

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