Introduction

Over the past decade, viral gene delivery has progressed from being merely an application in animal research to becoming an experimental therapeutic strategy in humans. Among the viral vectors, adeno-associated virus (AAV) serotypes have been identified as a promising means for gene delivery because they have important advantages over other vectors: they do not exhibit pathogenicity in humans and they provide significantly longer transgene expression.¹

Numerous AAV serotypes have been identified with variable tropism; however all of them share some properties including genome size and organization and, with small differences, the inverted terminal repeats share structure and function. All the serotypes are non-enveloped, single-stranded DNA parvoviruses, 25 nm in diameter, belonging to the genus Dependovirus. The upstream open reading frame encodes four replication (rep) proteins that allow AAV2 rep-proteins to package AAV2 inverted terminal repeat–flanked transgenes into nearly all serotype virions. The recombinant (r) forms of wild-type AAV serotypes retain the ability to traffic to the nucleus, uncoat and transduce cells, and their genomes become episomes or integrated proviruses.^2,3,4,5

The overall level of amino acid identity in the capsid protein of AAV serotypes 1–9 is 45% (refs. 6,7,8,9,10), with the most divergent serotypes being AAV4 and AAV5. Interestingly, the variability between serotypes is not evenly distributed throughout the sequence of the capsid protein, but is concentrated in the looped out domains that are displayed on the surface.¹¹ It has not yet been determined whether the variable domains are responsible for the differences in the expression levels and duration of expression seen in in vivo comparative studies of AAV serotype gene expression.

The greatest amount of in vivo transgene expression has been generated using AAV2. Comparative tropism evaluations of AAV serotypes have been examined in a number of studies: in the retina using AAV1–5 (ref. 12); in the brain using serotypes 1, 2, and 5 (ref. 13), 7–9, and rh10 (a recently described primate serotype),^9,14 1,2,5,7, and 8 (ref. 15), and 2,5,8, and rh10 (ref. 16), 8, 9, Rh10, and Rh43 (ref. 17); in vascular tissue;^18,19 in the lung;^20,21 in the liver;^22,23,24,25 in skeletal muscle;^26,27,28 and in cardiac muscle.^{29,30,31,32,33} In all these studies, the tissue distribution of AAV serotypes has been shown to differ depending on the route of delivery. Therefore a comparison between serotypes that have been delivered through different routes will be difficult to undertake. In order to better compare serotype vector properties, the same route of delivery should be used, and evaluations should include kinetics, level and persistence of expression and anatomical localization of the delivered transgenes. Nevertheless, for the majority of markers or therapeutic genes it is difficult to determine all these parameters in the individual animal over time.

These limitations are solved using marker genes for which the expression can be imaged at multiple time-points, thereby allowing for longitudinal studies in each animal. Using luciferase as a transgene, the activity of the transgenic protein can be evaluated after intraperitoneal injection of its substrate (luciferin), and the measurements can be performed sequentially in the same animal.^34,35,36 The advantages of this approach are: (i) it is unnecessary to kill animals at each time-point, and this reduces the requirement for animals; (ii) kinetic data can be obtained from each animal in the study, and (iii) individual variations in expression for the same serotype can be evaluated.

The overall purpose of this study was to investigate the tropism and kinetics of expression for nine different serotypes of AAV, packaging the same transgene, produced and purified in the same manner, and injected through the same systemic route. Here we report transgene expression from mice followed sequentially for 100 days, and then at 9 months after tail vein (TV) delivery of the luciferase transgene by AAV serotypes 1–9. We performed imaging and correlated it with postmortem protein levels and vector genome copies. The results give a broad insight into how AAV serotypes profoundly differ in their ability to transduce organs, and hopefully the data will help guide the use of each AAV serotype in future studies.

Results

Study design

This is a comparative study of AAV serotypes 1–9–mediated transgene expression after systemic delivery by TV injection. The capsid genes of the serotypes were pseudotyped by cloning into helper plasmids containing the AAV2 rep gene. The packaging vector contained the Luciferase gene cDNA driven by a cytomegalovirus (CMV) immediate-early promoter/enhancer flanked by AAV2 inverted terminal repeat sequences (Figure 1a). The TV route was chosen so as to assess AAV serotype tissue tropism; the luciferase transgene was used for visualizing the relative vector distribution in all the animals in a real-time manner, and for determining serotype expression hierarchies. Ventral and dorsal images were examined at each time-point. Animals injected with each of the serotypes (n = 4 or 5) were imaged at 7, 14, 29, 56, and 100 days post-injection (dpi). In addition, at least one animal from each serotype was imaged at 9 months after the injection (Figure 1b). At 100 dpi, the animals were killed and several of the organs were dissected, including brain, kidney, heart, liver, lung, skeletal muscles (gastrocnemius, hamstring, quadriceps), and testes. From these organs, proteins and DNA were examined for luciferase expression and viral genome copy number, respectively.

Figure 1.

Study design. (a) Diagram of packaging vector containing AAV2-ITRs and the cytomegalovirus (CMV)-driven luciferase transgene. This transgene was packaged within adeno-associated virus (AAV) serotypes 1–9. (b) After tail vein injection, the mice were imaged according to the above schedule. Most of the mice were killed at 100 days post injection; however, some mice were imaged up to 9 months after the injection. ITR, inverted terminal repeat.

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Onset of expression

Onset of expression is the time-point at which the light released by luciferase activity is distinguishable from background. After injecting 1 10¹¹ vg of AAV serotypes 1–9 into the TV of Balb/C mice, we observed expression of AAV serotypes 1, 6, 7, 8, and 9 at 7 dpi, which indicates that expression began prior to that time-point (data not shown). AAV4- and 5-injected animals first displayed luciferase activity at 14 dpi (data not shown), while AAV2 and 3 did not have observable expression until 29 dpi (other than in the tail; Figure 3a).

Figure 3.

In vivo imaging of luciferase after tail vein (TV) injection of adeno-associated virus (AAV) serotypes 1–9. (a) Low-expression group. For AAV serotypes 2–5 the expression range was 5,000–25,000 photons/second/cm². The images are shown at 29, 56, and 100 days post-injection (dpi). In order to discount AAV3 expression in the tail, a mask was placed over the tail in the last image. (b) Medium-expression group including AAV 1, 6, and 8. In this group, the range of expression was 15,000–200,000 photons/second/cm². (c) High-expression group: for AAV7 and 9 the expression range was 10,000–1,000,000 photons/second/cm². In both medium- and high-expression groups the images are shown at 14, 29, 56, and 100 dpi in a ventral view, and at 100 dpi in a dorsal view (bottom row). (d) Luciferase detection at 9 months after TV injection of AAV1–9.

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In vivo levels and kinetics of luciferase expression

A time-course plot was used for analyzing the levels of expression in animals injected with different AAV serotypes. We evaluated the total number of photons/second/cm²/radian (total flux, TF) emitted from the ventral images of each mouse at 7, 14, 29, 56, and 100 dpi, and at 9 months after the injection, using the Xenogen IVIS100 imaging system (Figure 2). The light emitted by these serotypes was in a wide range. On this basis, we categorized animals injected with AAV2, 3, 4, and 5 in the low expression group; AAV1, 6, and 8 in the medium group; and AAV7 and 9 in the high expression group. In the low-expression group, AAV2-injected animals showed a mean peak expression at 100 dpi (5.2 10⁵ TF), which declined at 9 months to 1.7 10⁵ TF. In contrast, AAV4- and 5-injected animals showed increased expression at the 9-month time-point (8.5 10⁵ TF for AAV4, and 1.3 10⁶ TF for AAV5) (Figure 2). AAV3 seemed to reach a plateau of 4.3 10⁵TF at 100 dpi, and this value held stable up to 9 months. In the medium-expression group, AAV1 and AAV6 showed a slow increase of expression up to 9 months (5.5 10⁶ TF for AAV1, and 1.2 10⁷ TF for AAV6), while the expression in AAV8-injected animals peaked at 56 dpi (1.3 10⁷ TF). In the high-expression group, the expression in AAV7-injected animals reached a plateau at 56 dpi (2.5 10⁷ TF), maintaining that level up to 9 months. AAV9-injected animals showed the maximum vector expression, reaching 7.6 10⁷ TF at 100 dpi and then declining up to 9 months (Figure 2).

Figure 2.

Kinetics of adeno-associated virus (AAV) serotype-mediated luciferase expression. Total flux (TF) (photons/sec/cm²/radian) released by luciferase activity at different time-points. For each serotype the mean flux was determined by averaging the flux emitted from each animal at each time-point. Luciferase expression from the tails of the animals was excluded while measuring TF. The values used are from the ventral images and are shown as mean values SEM.

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Localization and intensity of luciferase expression

In the low-expression group AAV2, 3, and 5 had similar localization patterns on the ventral axis. These animals showed bioluminescence related to vector expression from the upper abdominal and hindlimb regions; however, AAV3 expression was not evident until the 9-month time-point (Figure 3a and d). In contrast, AAV4 expression was localized to the thoracic cavity with no bioluminescence from the upper abdominal cavity (Figure 3a). However, by 9 months after the injection, some expression was found localized to the lower left side and hindlimbs (Figure 3d). Little expression was found localized to the dorsal axis in these animals (data not shown). In the medium-expression group, AAV1 and 6 induced overlapping patterns of expression with different biases, while AAV8 has a unique expression pattern. AAV1 and 6 induced expression in the thorax, abdomen, and hindlimb in the ventral axis and in the hindquarter muscle in the dorsal axis. AAV1 seemed to have a bias toward the upper abdomen, whereas AAV6 displayed a bias for the thoracic cavity (Figure 3b). The localization of expression persisted for 9 months (AAV1 and 6) as seen in Figure 3d. Evidence of expression in AAV8 injected animals is seen in the hindlimb, and also in the lower abdomen and upper thorax as seen in Figure 3b at the time-point of 56 dpi. This broad pattern of expression is maintained at 9 months after the injection (Figure 3d). AAV7- and 9-injected animals displayed the most intense expression with similar patterns. Both serotypes showed strong localization to the upper abdomen, with AAV9 having a more general distribution of expression throughout the body (Figure 3c). However, in contrast to AAV9, AAV7 did not express in the upper thorax and head. Additionally, AAV9-injected animals showed strong expression in the dorsal hindquarter (Figure 3c). Nine months after the injection, the pattern and distribution of expression in all the animals was seen to be maintained (Figure 3d).

Luciferase protein expression in selected tissues

In order to further analyze the transduction efficiency of the different AAV serotypes, we examined the luciferase enzyme activity in various tissues (Figure 4). In the low-expression group, the level of luciferase enzyme activity in examined tissues confirmed the patterns of localization observed during imaging (Figure 3a). AAV2 had its most intense expression in the liver tissue [545 194 relative light units (RLU)/mg total protein], this level of transduction being 26 and 30 times greater than the levels observed in hamstring and heart, respectively. The main localization of AAV4 expression was in the heart (2,373 635 RLU/mg total protein), followed by the lung (1,288 43 RLU/mg total protein), while AAV5 expression was low and localized only to the liver tissue (433 142 RLU/mg total protein). In this group, AAV3 showed the lowest expression levels, mainly in the heart (81 14 RLU/mg total protein) and liver (73 11 RLU/mg total protein). In the medium-expression group, AAV1 was expressed mainly in the liver (243 67 RLU/mg total protein), followed by heart and hindlimb skeletal muscles. AAV6 showed high enzyme activity levels in the heart (6,570 234 RLU/mg total protein) and this transduction was 2.7- and 4.7-fold greater than the levels observed in the gastrocnemius muscle and the liver, respectively (Figure 4). Although AAV6-mediated luciferase protein was also observed in the lung, the level was 10 times lower than the expression level in the heart. AAV8-mediated luciferase expression was localized mainly to the heart (4,175 1,311 RLU/mg total protein), followed by gastrocnemius, hamstring, and liver. Within the high-expression group, AAV7- and AAV9-mediated luciferase enzyme activity was found mainly in the liver (18,527 2,289 and 18,550 2,746 RLU/mg total protein, respectively). Both these serotypes showed high luciferase enzyme activity in the hindlimb skeletal muscle and, in addition, AAV9 had the highest expression in the heart (11,201 1,423 RLU/mg total protein) as compared to the level of AAV7 (1,837 746 RLU/mg total protein) (Figure 4). In general, we observed a direct relationship between the luciferase protein expression (in RLU/mg total protein) in various tissues and the bioluminescent light patterns observed on imaging the animals using the IVIS system (see Figures 3 and 4).

Figure 4.

Luciferase protein expression profile of adeno-associated virus (AAV) serotypes 1–9. The levels of luciferase activity [in relative light units (RLU) per mg protein] were determined in selected tissue at 100 days after intravenous injection of 1 10¹¹ particles of AAV1–9 into adult mice. The data are presented as mean values SEM.

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Examination of viral genome copy number remaining in selected tissues

As shown in Table 1, we compared the viral genome copy number remaining in the blood of mice injected with AAV serotypes 1, 4, and 6–9. The results show that each of the examined serotypes was associated with rapid clearance of viral genomes, with AAV1 and 4 showing rapid decrease within the first hour, AAV6–8 showing more significant drops in viral load between 1 and 6 hours, and AAV9 showing the slowest clearance (Table 1). Finally, at the 48-hour time-point, the amount of viral genomes remaining in the blood was <1% of the starting material for each of the serotypes examined.

Table 1 - AAV serotype clearance from blood after tail vein injection.

Full table (24K)

We next compared the viral genome copy numbers remaining in tissue samples at 100 dpi, using real-time-PCR (Figure 5). In the low-expression group, the AAV3 viral genome copy number did not exceed 10⁴/g in any tissue examined, and AAV2 exceeded 10⁴ copies only in the gastrocnemius and the kidney. AAV5 exceeded 10⁴ copies in the brain, kidney, and lung. In contrast, AAV4 exceeded 10⁴ copies in the kidney, brain, and gastrocnemius, and showed >10⁵ copies in the heart and >10⁶ copies in the lung (Figure 5). In the medium-expression group, AAV1 did not exceed 10⁴ copies in any tissue examined, the AAV6 copy number was >10⁴ in the liver and brain and >10⁵ in the heart, and AAV8 showed >10⁴ copies in the gastrocnemius, kidney, brain, and hamstring, and >10⁵ in the liver and lung (Figure 5). In the high-expression group, AAV7 showed >10⁴ copies in the kidney, hamstring, gastrocnemius, and testes and >10⁶ copies in the liver. AAV9 showed >10⁴ copies in all the tissues examined except testes and lung, with >10⁵ in the heart and >10⁶ in the liver (Figure 5).

Figure 5.

Vector genome copy numbers in selected tissues. Luciferase genome copy numbers/g of genomic DNA. Persistence of viral genomes in selected tissues 100 days after tail vein injection of 1 10¹¹ particles of adeno-associated virus (AAV) serotypes 1–9. Genomic DNA was isolated from the indicated tissues and 100 ng of each was used in triplicate to determine vector genome copies. Levels of significance were determined using one-way analysis of variance. The data are shown as mean values SEM. ^*P < 0.05 versus AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8. ^#P < 0.05 versus all. ^**P < 0.05 versus all.

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Determining the effect of viral transduction on heart function using Echocardiography

Because we observed a robust cardiac localization for several serotypes, transthoracic echocardiography was performed to evaluate whether cardiac function is affected by AAV transduction. As shown in Table 2, none of the AAV serotypes tested had any effect on cardiac function 100 days after TV injection. Specifically, the echocardiographic studies showed no significant differences in % fractional shortening among all the experimental groups and the control group (saline-injected). Additionally, left ventricular (LV) wall thickness and dimensions did not differ. We conclude that none of the serotypes tested had any effect on cardiac function after TV injection.

Table 2 - Echocardiographic parameters after tail vein injection of AAV 1-9 serotypes.

Full table (52K)

Discussion

Gene therapy has great potential for the treatment of a variety of diseases for which no definite pharmacological agents currently exist, such as heart failure, muscular dystrophies, and cystic fibrosis. Unlike other treatments, gene therapy aims to target the various underlying cellular and molecular abnormalities responsible for the disease. However, delivery of the gene of interest to the tissue poses a great challenge. In recent years, the gene therapy field has focused on AAV vectors because of their nonpathogenicity in humans, and their sustained and long-lasting expression after delivery. From clinical and therapeutic points of view, it is essential to know the tissue distribution and the kinetics (onset, duration of expression, and elimination) of AAV vectors. For this purpose, we sought to investigate the tissue tropism of various AAV serotypes after TV injection in mice. A major limitation of earlier studies was that the lack of uniformity in helper plasmid design, production, purification, and route of administration made it difficult to compare transduction efficiencies. In order to control for variability in these parameters, we standardized plasmid design, transfection, purification, tittering, dialysis, storage, and injection for all the serotypes examined. Consequently, the differences in transduction efficiencies and biodistribution profiles exclusively reflect the differences in the virion shells of these serotypes.

We chose TV injection because it allows the serotype tissue-tropism to be investigated. This route of administration is realistic therapeutically and it can still allow tissue-specific expression if tissue-specific promoters are used in concert with vector administration. Importantly, we report for the first time the differences in tropism and kinetics of nine commonly used AAV serotypes, and the data from the examination of vector expressions for as long as 9 months after TV injection. We examined the clearance of viral genomes from the blood within the first 48 hours after TV injection. The findings of the study suggest that the majority of AAV1 and 4 virions are removed from the blood within the first hour, AAV6–8 is removed more slowly (between 1 and 6 hours), while AAV9 is removed between 6 and 48 hours after the injection. When directly comparing the rate of AAV clearance from blood with tissue expression, we could not see any predictive power of the blood clearance on kinetics, biodistribution, or tropism exhibited by the AAV serotypes. The luciferase images ran parallel to quantitative assays of protein concentration and DNA content, making this a useful method for examining additional serotypes. The exception to this was AAV1; images show moderate levels of expression in the upper abdominal region and in the hindlimbs, whereas the luciferase enzyme activity and genome copy numbers in these areas were very low. This suggests that the genomes present in the liver and hindlimbs are expressing more efficiently. Another exception was AAV4 expression in the thoracic cavity which put it in the low-expression group, whereas the genome copy number for AAV4 was the highest of all serotypes in the lung and among the highest in heart. Two possible explanations for the low visual expression are (i) the chest cavity is surrounded by muscle and bone which is a barrier to light emission and (ii) uncoating in the case of AAV4 is slower than in other serotypes.⁵ The observed high levels of RLU in the lung and heart support the first explanation in the case of AAV4.

The most common organ transduced was the liver; each serotype with the exception of AAV4 demonstrated some upper abdominal expression. High protein levels were observed with AAV9, 8, 7, 6, 1 and, to a lesser extent, with 5 and 2. AAV8 expression was visually more uniform throughout the hindlimb, abdominal, and thoracic regions. Wang et al.,²⁸ using intraperitoneal injection of neonatal mice, demonstrated the widespread expression profile of AAV8 green fluorescent protein. Inagaki et al.²⁴ injected AAV8 and 9 through the TV in a dose escalation study and observed high vector genome copy numbers for both serotypes in the liver. Our data is consistent with those of Inagaki et al.²⁴ for AAV9 genome copy numbers. Several studies have shown minimal differences in peripheral and mesenteric-directed liver delivery. Nathwani et al.³⁷ reported 44 diploid vector genomes/hepatocytes 1 month after peripheral vein injection while 68 diploid genomes were seen after liver-directed delivery for AAV8 at 1 10¹² vg/kg in rhesus monkey, a difference of 35% . Nakai et al.²⁵ observed that AAV8-mediated EF1a-nuclear localized lacZ expression in hepatocytes was approximately equivalent irrespective of the route of administration, being 24% when using the portal vein route and 15% when using the TV for injecting 3 10¹¹ vg/total in mouse, again a difference of 35% . Sarkar et al.,³⁸ using AAV8, showed that the TV and portal vein injection routes were equally efficient in correcting factor VIII deficiency in mice. This is in contrast to the findings with other serotypes, wherein it has been reported that transduction is more efficient with direct liver delivery than with TV.³⁹ Although the difference between systemic and tissue-directed delivery approaches are important, this study was designed to examine biodistribution after peripheral delivery of AAV1–9.

Hindlimb skeletal muscle was the next most common region showing expression after TV injection of serotypes AAV1–9. Additionally, AAV serotypes 1, 6, and 9 showed high levels of luciferase expression in the dorsal hindquarter muscles. It is unclear why the hindlimb skeletal muscle is targeted by nearly all the serotypes tested; a potential explanation could be that the hindlimb skeletal muscles are sites of high-level metabolic activity and increased blood flow. Several studies have demonstrated hindlimb skeletal muscle transduction by various serotypes including, AAV6 (ref. 30), AAV1, 6, and 8 (ref. 28), and AAV8 and 9 (ref. 24) after TV injection in mice.

The heart was transduced by several serotypes including AAV4, 6, 7, 8, and 9, with AAV9 having the highest luciferase expression and viral genome copies. Transgene expression mediated by AAV9 transduction was greater than those by AAV4 (4.7), AAV8 (2.7), and AAV6 (1.7) in the heart after TV injection. We clearly saw thoracic expression of AAV4 and 6, while the expression is masked for AAV9, potentially because of the intensity of upper abdominal expression. Recently, several publications have described cardiac transduction after systemic administration of AAV serotypes. Gregorevic et al.,³⁰ using AAV6 TV injection, showed greater transgene expression and vector genome copy numbers retained in the heart as the level of exogenous vascular endothelial growth factor increased. Inagaki et al.²⁴ demonstrated higher transduction efficiency and accumulation of viral genome copies in the heart in a dose escalation series comparing AAV9 with AAV8. For AAV9, they showed <1 viral genome/cell at 1 10¹¹ total particles injected and this is approximately the same number of viral genomes that we isolated from the heart (Figure 5). Müller et al.³¹ used TV injection to analyze the transduction efficiencies of AAV1–6, carrying the CMV-driven luciferase transgene. Their results demonstrated higher levels of luciferase activity in the heart with AAV 4 and 5 than with AAV6 at 3 weeks after injection of nmRI female mice. This is in contrast to our observation, where AAV6 was superior to AAV4 and 5 at 100 dpi. These differences may result from the variations in the strain or gender characteristics of the mice.

It is extremely important to understand the tropism of AAV serotypes in mouse models. This is because model therapies with specific targets are required in order to assess potential primary and secondary viral targets. Such an understanding will also prove useful when comparing with larger animal models that will be used before any future human applications. In this study, the transduction efficiencies of AAV serotypes 1–9 were examined serially, using bioluminescence imaging over a 9-month period. We have shown that the kinetics of expression vary greatly between these serotypes, with AAV7 and 9 having the fastest onset of expression, and AAV3 and 4 the slowest. Further, our results demonstrate that the primary targets for AAV serotypes 1, 2, 5, 6, 7, and 9 are the liver and hindlimbs. AAV8 and 9 transduce tissues more ubiquitously than the other serotypes do, with AAV9 having the most robust tissue expression. AAV9 showed low levels of luciferase protein expression and genome copy numbers in the brain and testes. The expression in the testes is an important safety issue for future clinical investigation of this serotype. AAV7 has a strong tropism for the liver, while AAV6 has some bias for the heart. AAV2, 3, 4, and 5 transduce with low efficiency. Surprisingly, AAV4 has a specific tropism for the lung and heart, with low transduction efficiency and viral genome copy numbers in the liver. These results will aid researchers in their choice of serotype for specific gene delivery applications, and will afford an important basis for comparison when these and other serotypes are used in large animal models.

Materials and Methods

Cell lines and plasmids. HEK 293 and HeLa cells were maintained at 37 °C with 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin–streptomycin. The AAV serotype–specific helper plasmids pXR1-9 contain their respective serotypes capsid proteins and package AAV2 inverted terminal repeats, as have been described earlier.⁵ The pXR6 and pXR8 plasmids were a gift from Jude Samulski. Portions of AAV7 and AAV9 capsid coding sequence (GenScript, Piscataway, NJ) were synthesized and cloned into pXR8 and pXR3, respectively. The plasmid pXX6-80, containing the genes necessary for adenovirus helper function in AAV replication, has been described elsewhere.⁴⁰ The transgene plasmid, pTRCMV-Luc, was constructed by digesting pgL2-basic vector (Promega, Madison, WI) and pTRUF3 with XhoI and SalI, and then ligating the 2,711-base pair fragment containing the firefly luciferase gene into the large vector fragment of pTRUF3 (Figure 1a).

Production of recombinant virus. The viruses were all produced by the triple transfection method, using HEK 293 cells.⁴⁰ HEK 293 cells were expanded to transfect 20–15-cm plates for each serotype. For each 15-cm plate, 1 10⁷ cells were plated the day before the polyethylene imine (Polysciences, Warrington, PA)-mediated transfection, using 7.5 g of pTRCMVLuc and pXR (1-9) and 15 g of pXX680. Seventy-two hours after transfection, the cells were collected by centrifugation and the cell pellet was resuspended in 19 ml ddH₂O on ice. Leupeptin was added to the resuspended cells before sonication (Branson Sonifier 250; West Chester, PA). Next, benzonase (Sigma, St. Louis, MO) was added to a final concentration of 0.5 U/l, before incubating at 37 °C for 60 minutes. Each serotype was subjected to two rounds of cesium chloride isopycnic gradient centrifugation to provide a uniform purification method. To each milliliter of solution 0.58 g of cesium chloride was added, and the mixture was centrifuged at 35,000 rpm for 48 hours. After the second centrifugation, the peak fractions, as determined by dot blot hybridization and transduction of HeLa cells, were dialyzed for three rounds against 1 phosphate-buffered saline containing 5% sorbitol. Viruses were stored at –80 °C and thawed on ice before commencing the experiment. The total vector genome number was determined by dot blot hybridization, with all serotypes titered by serial dilution (12.5, 6.25, 3.125, 1.56, and 0.78 l) in duplicate. AAV4 and 9 consistently gave the highest titer, while AAV7 gave the lowest. AAV1 Luc 4.2 10¹¹/ml, AAV2 Luc 4.2 10¹¹/ml, AAV3 Luc 1.5 10¹²/ml, AAV4 Luc 9.2 10¹¹/ml, AAV5 Luc 8.5 10¹¹/ml, AAV6 Luc 1.7 10¹²/ml, AAV7 Luc 3.2 10¹¹/ml, AAV8 Luc 1.5 10¹²/ml, and AAV9 Luc 6.0 10¹²/ml.

Procedures involving animals: All the experiments involving animals were conducted in accordance with the Institutional Animal Care and Use Committee of Thomas Jefferson University. Male Balb/C mice were purchased from Charles River Breeding Laboratories (Wilmington, MA). At 8–10 weeks of age the animals were injected in the TV, with 1 10¹¹ particles of AAV-CMV-luciferase in 312 l of 1 phosphate-buffered saline supplemented with 5% sorbitol. It was ensured that the injection administered for of each of the serotypes was of the same volume as the one used for the serotype with the lowest titer. Control animals were injected with 312 l of 1 phosphate-buffered saline supplemented with 5% sorbitol. The animals were held in the Tail Veiner TV-150 position (Braintree Scientific, Braintree, MA), and their tails were warmed before the injection. The injections into the veins were carried out using 28 gauge needles. All the mice recovered from the injection quickly without loss of mobility or interruption of grooming activity.

In vivo animal imaging. The mice were anesthetized with 2% isofluorane and oxygen. The D-luciferin substrate (Biotium, Hayward, CA) was injected intraperitoneally, at a dose of 150 g/g of body weight. The mice were then placed in a light-tight chamber, and images were generated using a cryogenically cooled charge-coupling device camera IVIS 100 (Xenogen, Alameda, CA). For each mouse, the images were taken at 8–12 minutes after the substrate injection. Light and dark images of mice were collected, and the dark images were pseudocolored using the Xenogen software. The visual output represents the number of photons emitted/second/cm² as a false color image where the maximum is red and the minimum is dark blue. All animals were imaged on a schedule of 7, 14, 29, 56, and 100 days after AAV vector injection; one set of animals was also imaged at 9 months.

Quantifying luciferase expression in mice. At each time-point a "region of interest" was designated surrounding each animal in order to quantify the TF (photons/sec/cm²/radian) being released by luciferase activity. For each serotype, the mean flux was determined for all the animals at each time-point.

Echocardiography. At 100 dpi, echocardiographic studies were performed on each animal using an ultrasonographic system (VEVO-770; VisualSonic, Toronto, Canada). After being anesthetized with 1.5% isofluorane, the 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 thicknesses were made using the leading-edge convention of the American Society of Echocardiography. The percentage of LV 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 protein activity. At 100 days after the TV injection of AAV-CMV-luciferase 1–9 serotypes, the animals were killed and the selected organs were dissected: brain, heart, lung, liver, kidney, testes, and skeletal muscles (gastrocnemius, hamstring, and quadriceps from both hindlimbs). Forty milligrams of each tissue was homogenized with 80 l of 5 passive lysis buffer (Promega, Madison, WI) and 320 l of ddH₂O. In order to examine the amount of Luciferase protein, 20 l of each homogenized tissue were aliquoted in triplicate in an opaque 96-well plate. A Wallac luminometer (Applied Biosystems, Foster city, CA) was used for capturing luminescence over a 10-second interval after the addition of 100 l of substrate (luciferase assay reagent, Promega, Madison, WI) for each well. A recombinant luciferase standard was added in triplicate at three different concentrations (1, 5, and 50 pg) to each plate as a means of quantifying expression (QuantiLum Recombinant Luciferase; Promega, Madison, WI). For tissue samples, the amount of luciferase protein activity in RLU was determined by normalizing the experimental samples to the standard. Total protein concentration was determined using a bicinchoninic acid assay kit (BioRad, Hercules, CA).

DNA isolation and real-time PCR. DNA isolation from tissues was performed using the Puragene kit (Gentra Systems, Minneapolis, MN) in accordance with the manufacturer's protocol. Total DNA concentration was determined by spectrophotometry using Nanodrop, and 100 ng of DNA from each sample was used as the template material for real-time-PCR. Real-time PCR was performed on each sample for both the luciferase gene in order to determine copies of the viral genome, and the mouse neural cell adhesion molecule intron sequence,⁴¹ to standardize for number of mouse genomes present in each sample.

The primers used for luciferase were: 5'-CAGGGATTTCAGTCGATGGTA-3'(forward) and 5'-GGACTCTGGTACAAAATCGTA-3'(reverse). The primers used for neural cell adhesion molecule were: 5'-CCCACGGTTTTCTTCCTACA-3' (forward) and 5'-AATGGCAAAGCTGGAATTTG-3' (reverse).

The accumulation of PCR products for each gene was measured using cybergreen. All the samples were run on an iCycler (Bio-Rad, Hercules, CA). Neural cell adhesion molecule plasmid DNA at 100 pg, 10 pg, and 1 pg, and Luciferase plasmid DNA at 10 pg, 1 pg, and 100 fg in 10 ng/l salmon sperm DNA were used on each real-time-PCR plate as copy number controls. All samples and controls were run in triplicate. From this data set, we obtained the number of viral genome copies/g of total DNA for each serotype. One microgram of mouse genomic DNA contains 3 10⁵ haploid genomes.

In order to determine the rate of viral clearance in blood, 1 10¹¹ viral genomes were injected by the TV route in the same volume that was used in the imaging experiments. At 1, 6, and 48 hours after the injection the animals were killed and 200 l of blood was drawn. Viral DNA was isolated form the blood in accordance with the protocol described in the high pure viral nucleic acid kit (Roche, Basel, Switzerland). For control samples, 200 l of blood was removed from animals injected with 312 l of 1 phosphate-buffered saline + 5% sorbitol. To this control blood, either known titers of virus from the same serotypes were added (positive control), or no virus was added (negative control). These samples were processed in accordance with the High Pure Viral Nucleic Acid protocol. Total DNA concentration was determined by spectrophotometry using Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE), and 100 ng of DNA from each sample was used as the template material for real-time-PCR.

Statistical analysis: We compared mean values from different experimental groups using a two-tailed Student's t-test or one-way analysis of variance.

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Acknowledgments

This work was supported by grants from the American Heart Association AHA F69103 (to J.E.R.), and by funding from the Pennsylvania Department of Health, Pennsylvania Tobacco Grant A75301.