Introduction

1-Antitrypsin (AAT) deficiency is a well-described single-gene defect for which muscle-directed recombinant adeno-associated virus (rAAV)-based gene therapy is already in clinical trials¹. One particular AAT mutation, PiZ homozygous deficiency, affects 1 in 6000–8000 births in the United States². AAT is a 52-kDa protease inhibitor predominantly expressed in the liver, from where it is secreted to act upon its major target organ, the lung. Within the lung, AAT helps prevent damage to the extracellular matrix and to cells by balancing the protease:antiprotease ratio. The primary substrate is neutrophil elastase (NE), which AAT binds to and inactivates. AAT has additional activity against cathepsin B and other proteases. When serum concentrations of AAT fall below 11 M (<570 g/ml)^3,4, the protective effect is lost and the uncontrolled activity of NE proceeds to destroy the elastin fibers supporting the alveolar network, leading to loss of airway tethering and destruction of alveolar septae. Current clinical treatments involve the infusion of purified protein or plasma-derived blood products^5,6,7. These lengthy treatments can be performed weekly with a significant protective effect and few side effects, but with considerable cost and discomfort to the patient. A subset of patients with AAT deficiency also experiences liver disease, which is due to accumulation of the PiZ mutant due to impaired secretion within hepatocytes.

For clinical replacement, gene therapy for lung disease in AAT deficiency can be viewed as a long-lasting alternative to protein replacement therapy, with the goal being maintenance of serum levels at or above the 11 M threshold. Delivery of the AAT gene (approved symbol SERPINA1) to any tissue capable of expressing and secreting this protein would be expected to accomplish this goal. Indeed, AAT expression and secretion have been demonstrated from a number of ectopic sites, including muscle^8,9, bronchial epithelium¹⁰, and skin¹¹. In contrast, any gene therapy with potential to target both liver and lung disease must involve delivery of therapeutic molecules to the hepatocytes themselves. Down-regulation of the mutant PiZ-AAT with ribozymes in vitro has been demonstrated by Zern et al. using an SV40-based vector¹², but any such approach will require a vector capable of transduction of a high percentage of hepatocytes. Standard rAAV2 vectors, such as those used in current clinical trials, appear to be incapable of stably transducing more than 5% of the hepatocyte population. Recombinant AAV2/8 pseudotyped (rAAV8) vectors have recently been shown to be more efficient for hepatocyte transduction than rAAV2 vectors¹³. In the current study, we evaluated a rAAV2/8-AAT vector to assess its potential for stable and efficient delivery of human AAT in the C57Bl/6 mouse. We also evaluated the percentage of hepatocytes transduced and the molecular fate of rAAV2/8-delivered genomes.

Results

In vivo liver transduction comparison of AAV serotypes

To continue to enhance transduction of liver hepatocytes for increased transgene expression, we evaluated the newly discovered AAV8 serotype with three rAAV serotypes used in our lab and by others. We packaged the human 1-antitrypsin complementary DNA sequence driven by the highly active cytomegalovirus (CMV) enhancer/chicken -actin promoter (CB) plasmid into virions by cotransfection with plasmids containing the capsid sequences for AAV serotypes 1, 2, 5, and 8. All ITRs were from serotype 2 to control for possible differences in genome manipulations once uncoated. We introduced all four vectors into C57Bl/6 mice via direct injection into the portal vein with 9.6 10¹⁰ particles per animal. We evaluated infection efficiency by analyzing serum levels of the AAT transgene by ELISA for up to 24 weeks. Peak levels for each vector were obtained within 1 to 3 weeks postinjection and remained stable throughout the course of the study (Fig. 1A). We saw a greater than 100-fold enhancement of hAAT by AAV2/8 over AAV2/2 and a 1000-fold advantage over AAV2/1 and 5.

Figure 1.

Transduction of hepatocytes by AAV serotype 8 produces high levels of 1-antitrypsin and more genome copies. (A) Packaging plasmids encoding the capsid sequences for AAV serotypes 1, 2, 5, and 8 were cotransfected with pCBAT into 293 cells to generate complete virions. The array of viruses generated was titered and injected into the portal vein of C57Bl/6 mice to target the liver with equal doses of 9.6 10¹⁰ vector particles per animal. At time points of 0, 1, 3, 5, 8, 12, 16, 20, and 24 weeks postinjection, serum samples were analyzed for transgene (AAT) expression, and means at each time point are graphed. A similar pattern of heightened expression by 1 week can be detected for each serotype and stable transgene levels are detected throughout the study. (B) Twenty-four weeks after being injected through the portal vein with 1 10¹¹ particles, animals underwent a partial hepatectomy. Liver biopsies were collected and genomic DNA was extracted. Purified DNA was assayed by quantitative RT-PCR for AAV copies using the pCBAT plasmid as the standard and using the 166,000 mouse genomes per microgram of genomic DNA standard. The mean standard deviation of copies per cell for each animal is plotted for comparison.

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To confirm AAV2/8 further as a more efficient liver-mediated gene therapy vector, we excised liver tissue at 24 weeks and subjected genomic DNA to real-time PCR. Primer probes specific for the CB promoter were used in a reaction with 1 g of genomic DNA. Copy number per cell correlated with hAAT transgene expression in terms of AAV2/8 outperforming vectors based on AAV serotypes 1, 2, and 5 (Fig. 1B). In two cases, though, animals injected with the AAV2/8 vector had genome counts per cell that were lower than the range for those receiving AAV2, but were still secreting hAAT levels high enough to contribute to the group's log increase average. The AAT levels from these two subjects may result from transduction and secretion from pancreatic cells, where positive staining was observed (data not shown). Also, a maximum concentration of circulating hAAT in the murine serum may have been reached. Therefore, the full potential of using AAV2/8 in the liver may not be seen. Overall, the higher AAT protein levels reflect the number of vector genomes per cell within the liver.

Histology and immunohistochemistry for human 1-antitrypsin in the murine liver

Administration of AAV vectors did not result in morphological changes as assessed on H&E-stained paraffin sections for all mice. Occasional focal to multifocal mononuclear infiltrates were observed in PBS- and vector-treated animals and were considered normal for animals from this colony.

We subsequently performed immunostaining of mouse liver sections for the human AAT protein to determine the proportion of hepatocytes infected. We confirmed specificity of the primary antibody for human AAT using normal human liver compared to normal mouse liver (data not shown). Additionally, visualization of the tissue allows for the determination of cell types transduced and intensity of AAT expression. As shown in Fig. 2A, we observed no staining for human AAT in a PBS-injected animal. In comparison, the AAV2 (Fig. 2C) and AAV2/1, 5, and 8 (Figs. 2B, 2D, and 2E, respectively) pseudotype-treated animals consistently show positive staining. Semiquantitative evaluation of human AAT immunoreactivity revealed a positive signal for each serotype for nearly all criteria in contrast to no staining in the PBS-injected controls. Observed trends included scores of "2" and above for only type 2- and 8-transduced tissue in the categories of panlobular blush, which can result from the AAT secreted being endocytosed by neighboring cells and midzonal and periportal staining. Only AAV2/8-injected liver tissue resulted in scores of "3" or above. Decile scores, an indicator of the overall percentage of AAT-positive cells with intensity over the blush level, were at least in the 0–10% range for every mouse scored in all pseudotype groups. Most impressively, only the AAV8 tissue reached decile scores up to 40%. Of note, additional positive cell types and patterns seen in this group include bile duct staining in seven of eight animals reviewed and intense nuclear and granular, diffuse cytoplasmic staining in AAV8-treated animals. The trends observed herein parallel those seen in genome copy number and circulating AAT levels with AAV2 immunoreactivity exceeding the AAV2/1 and 2/5 groups and AAV2/8 outperforming all groups in cellular intensity and positive spread over the tissue.

Figure 2.

Immunostaining for hAAT in the murine liver. All sections were incubated with a rabbit anti-human AAT antibody as the primary antibody. (A) Liver section of a PBS-injected mouse to serve as a negative control reference showing no positive staining for hAAT. (B) AAV2/1-CBAT, (C) AAV2-CBAT, (D) AAV5-CBAT, and (E) AAV8-CBAT liver sections from mice administered with 1 10¹³ vector particles via the portal vein. Staining of livers from each serotype reveals an overall panlobular blush, midzonal, periportal, and perivenous detection of hAAT. Central veins (CV) and portal veins (PV) are noted. (F) 40 magnification of outlined portion in (E) demonstrating staining around the central vein. Tissues were harvested at 24 weeks postinjection during a partial hepatectomy procedure. Original magnifications: A–E, 20; F, 40.

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Persistence of AAV2/8 genomes in the liver is similar to that of AAV2 genomes

To determine the preferred molecular state of AAV2/8 in transduced liver cells, we performed a partial hepatectomy at 24 weeks in the previous group of animals. We observed the effects on transgene expression and molecular persistence after the partial hepatectomy by transgene expression, copies per liver cell, and Southern blot to determine molecular state. As shown in Fig. 3A, hAAT levels expressed from the various AAV vectors were plotted in relationship to pre-partial hepatectomy levels. As expected from previous studies¹⁷, AAV2 animals recorded a drop to 10 to 40% of pretreatment levels after a washout period of circulating AAT (compare week 1 to week 3). Likewise, cohorts injected with AAV2/1 and 2/8 saw an even greater decrease in hAAT to below 10% of week 24 values. Unexpectedly, AAV2/5 showed a decidedly different pattern with hAAT levels rising as liver regeneration occurred. Also, it is important to note that although administration was directed to the liver through an intravascular injection, one cannot rule out the possibility that the total AAT level determined in serum sampling is not in part due to expression from additionally infected organs and may be reflected in post-partial hepatectomy AAT level differences between pseudotype groups (Fig. 3A). For instance, AAV5 has been shown to have a greater affinity for pancreatic cells²¹ and the lung epithelial cells²². Although AAV2/5 is shown, this study specifically addressed serotype 8 in hepatocytes. The reduction in serum AAT data was supported by real-time PCR of liver genomic DNA upon sacrifice at 8 weeks post-partial hepatectomy. A direct correlation (r² = 0.9809) between the percentage decrease in hAAT (Fig. 3A) expression levels and percentage reduction in copy number is observed (Fig. 3B) before and after surgery. Similar to Fig. 3A, pseudotypes 1, 2, and 8 all exhibited a considerable loss of vector copies per cell after partial hepatectomy.

Figure 3.

Decreased levels of hAAT and AAV genome copies after partial hepatectomy for serotypes 1, 2, and 8. (A) At 24 weeks after portal vein injection, the animals in Fig. 2 underwent a partial hepatectomy that could remove up to 85% of the total liver weight. Starting 1 week after surgery serum sampling resumed until week 8, when the animals were sacrificed. AAT transgene means for each serotype cohort determined by ELISA are plotted as a percentage of the AAT levels obtained at week 24. A minor decrease at week 1 reflects the 2-week half-life of AAT in circulation. The increase in levels for the serotype 5 group may reflect a propensity toward integration compared to other serotypes. (B) This graph represents the AAV copy number as detected by real-time PCR before and after surgery removing three lobes of the liver. Each bar representing the mean and standard deviation of a serotype cohort is a percentage of the total copy number obtained the same way before surgery 8 weeks prior. Again, a trend toward copy regeneration within the proliferating liver is seen as well as the expected drop in copies for AAV8.

Full figure and legend (87K)

Both sets of data suggest AAV2/8 and AAV2/1 persist in a nonintegrated state similar to that of AAV2. One can infer episomal persistence because, with integration of the vector genome, liver regeneration would cause additional copies to be replicated during cell division. Unexpectedly, in both sets of experiments, pseudotyped AAV5-injected animals produced very different results from those anticipated. By 8 weeks after partial hepatectomy, AAT levels in these mice had returned to presurgical levels. In addition, quantification of total AAV genome copies before and after surgery resulted in nearly equivalent copy numbers at both time points. At least 90% of the copies present before removal of the liver were there at sacrifice, suggesting that some greater degree of vector genomes were integrated and replicated alongside liver regeneration. Support for this conclusion is the observation that the AAV2/5 group had an initial drop in AAT levels at week 1 after partial hepatectomy, thus indicating that productively transduced cells were removed along with the AAV genomes driving AAT expression. The wild-type AAV2/5 genome tends to differ from other known serotypes on several levels. These differences include an efficient transcription initiation start site within the ITR; all Rep-coding transcripts polyadenylate within the intron and adenovirus co-infection does not enhance Cap protein expression²³. Although significant, these differences and others not mentioned cannot explain the results presented in this study since all vectors were pseudotyped with AAV2 ITRs and genome sequences were identical. Therefore, the capsid proteins were the only difference and could result in a cellular processing that is different from that of the other three. Furthermore, the use of an endosomal pathway could potentially "mark" the DNA that leads to greater integration efficiency once in the cell. We can also try to explain this by the novelty of the partial hepatectomy procedure.

Molecular fate of the various AAV serotypes by southern blot

Another method of comparing the molecular fate of AAV serotypes whether episomal or integrated is by analysis of genomic DNA. To perform this, we purified total liver genomic DNA from the above-described experiment and digested the resulting DNA with an enzyme that either cuts once within the CBAT vector or cuts twice (Fig. 4A). We separated the digested DNA by agarose gel and blotted it to a membrane. A hAAT ³²P-labeled probe allowed for the identification and size determination of the restricted DNA (Fig. 4B). As previously observed and reported²⁴, AAV2 primarily exists long term in an extrachromosomal form, linear or circular, evidenced by the one-cutter digest bands running at a lower molecular weight than if the majority of the vector were integrated, for which a higher molecular weight band would appear (lane 11). In addition, it can be concluded that alternative serotypes 1, 5, and 8 persist in the same manner (lanes 2, 5, 7, 9, and 13). Furthermore, these data support the correlation of serum hAAT levels with cellular infection. After digestion with an enzyme that cuts twice within the vector, all AAV genome copies, whether integrated or episomal, will result in a 2.28-kb band representing the total cellular copies (lanes 3, 4, 6, 8, 10, and 12). An overabundance of genomes present in AAV2/8-treated animals is clearly shown in signal strength compared to AAV1, 2, and 5 pseudotyped vectors.

Figure 4.

Southern blot for genomic persistence of AAV serotypes in the liver. Total cellular genomic DNA was extracted from frozen liver tissue. Forty micrograms of genomic DNA was digested with either HindIII (one-cutter within the vector) or ApaI (two-cutter within the vector) overnight and separated on agarose gel. (A) Schematic of the restriction sites and expected band sizes and annealing location of the hAAT probe. (B) After blotting to nitrocellulose, a ³²P-labeled probe to the hAAT sequence was hybridized. Endogenous bands not vector related, head-to-tail (H-T) concatemers, and the double-digested internal fragments are noted.

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Dose response of AAV2/8 in vivo

While developing a viral vector for a secreted protein disorder such as AAT deficiency, the minimum effective dose may be sought for preventing toxic levels of the transgene and reducing production costs passed down to the patient. Therefore, we next conducted a dose escalation study with AAV8 to determine the scalability of this vector. We injected cohorts of C57Bl/6 mice with increasing doses of our AAV2/8/CBAT vector (1.65 10⁹, 1.65 10¹⁰, and 1.65 10¹¹ vector genomes/mouse) into the portal vein and measured the reporter protein expression by ELISA (Fig. 5). A 15-fold increase in transgene expression could be seen for each 10-fold increase of vector administered. This measure was maintained for the 24-week study. At the highest dose, maximum expression peaked at upward of 30 mg hAAT/ml serum. Interestingly, vector genomes (Table 1) established by real-time PCR show a different result. In this experiment, the medium- and high-dose groups had an average of 6.74 and 5.38 genome copies per cell, respectively. Although not significant, we may be seeing a case of receptor saturation upon vector administration. The additional increase in AAT levels in the high-dose group over the medium-dose group could be due to secreted AAT from lung and pancreatic tissue. In fact, after further examination of sectioned organ staining for AAT, acinar and islet cells within the pancreas stained positive (data not shown) for AAT.

Figure 5.

Dose response in AAV8-injected mice. High titer AAV8/CBAT virus was produced and injected via the portal vein into C57Bl/6 mice to determine a dose–response relationship. Three doses of AAV8, 1.65 10⁹ (low, N = 3), 1.65 10¹⁰ (medium, N = 5), and 1.65 10¹¹ (high, N = 4), were injected and serum AAT levels determined at weeks 0, 1, 3, 5, 8, 12, 16, 20, and 24. Means standard deviations are plotted on a log scale. An approximately 50-fold increase for each 10-fold higher dose of virus is observed.

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TABLE 1 - AAV genome counts do not reflect transgene expression.

Full table

A saturating dose does not affect molecular fate of AAV8

As stated above, a lack of random integration into the human genome in treated patients is a desired attribute when using AAV gene transfer. We next wanted to model the effects of using an increasing viral load in vivo by reaffirming the state at which AAV8 resides in the nucleus of infected cells. We investigated this by performing a partial hepatectomy in the medium- and high-dose animals used above and looking at transgene expression and the AAV genome state by Southern blot. Each mouse had three of four liver lobes removed to stimulate liver regeneration. Beginning with transgene expression, we analyzed serial serum samples for hAAT expression and plotted the results (Fig. 6). By 2 weeks, the lowest percentage AAT protein compared to pretreatment levels, 22% for medium- and 1% for high-dose cohorts, were reached and maintained for 8 weeks. The lack of reconstituted AAT levels is similar to that seen in the AAV serotype comparison study presented previously and suggests the vector genomes are not being replicated during regeneration. To confirm this, genomic copies of vector DNA at partial hepatectomy and sacrifice are compared in Fig. 7 and show a similar fall in total copies between each dose group.

Figure 6.

Demonstration of AAV serotype 8 genome persistence as episomes. The previously used AAV8 dose–response animals were subjected to a partial hepatectomy. hAAT levels present in the serum were quantitated by ELISA. The resulting data were plotted on a weekly timeline for visualization after control means were subtracted. The previously used "low dose" AAV8 group is not shown because of low levels difficult to distinguish from controls after partial hepatectomy.

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Figure 7.

Total AAV copy number decreases after partial hepatectomy in mice administered three doses of AAV2/8. This graph represents the AAV copy number as detected by real-time PCR before and after surgery removing three lobes of the liver. Each bar represents the mean and standard deviation of a serotype cohort as a percentage of the total copy number obtained the same way before surgery 8 weeks prior. A comparable reduction in total copy is seen with each dose group.

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Discussion

In the past few years, research using rAAV vector has focused on the enhancement of vector attributes rather than the biology of the underlying virus. This is in part because of the well-established safety profile seen in many long-term studies. Our goal for this particular study was to target liver hepatocytes through alternative serotypes to enhance the number of successfully transduced hepatocytes for the treatment of PiZ 1-antitrypsin deficiency. We compared three commonly investigated serotypes with the newly discovered AAV8 serotype isolated from rhesus monkeys¹³. After portal vein introduction into C57Bl/6 mice, serum AAT levels in the AAV8 vector-treated animals were significantly higher than for all other serotypes tested, up to 100-fold greater than for the AAV2 vectors. Similar results have been documented by other investigators¹³. The performance of AAV8 was also evaluated on the molecular level with real-time PCR for vector genomes and Southern blotting. Based on the finding of 166,000 double-stranded DNA genomes per microgram of isolated genomic DNA, an average of 4.3 genomes were present per cell in AAV8-infected livers, in contrast to 0.54 AAV genomes per cell for AAV2 tissue. Both sets of data confirm AAV8 as a better alternative to previously used serotypes for human AAT secretion from the liver. Finally, immunohistochemistry for AAT in liver sections of the injected mice showed up to 40% of the cell population was intensely positive for AAV8-treated animals. The difference may reflect the rate of uncoating of the capsids once internalized²⁵. In addition, the AAV8 capsid proteins may target internalized virions to an alternative endosomal pathway that leads to an increase in stable genomes. Of note, in this study AAV2/5 pseudotyped vectors behaved differently from 1, 2, and 8 pseudotyped vectors. After partial hepatectomy, regeneration of vector genomes was observed by copy number and transgene levels as the liver mass increased in size over time. Unfortunately, these results could not be confirmed by Southern blot analysis due to the low intensity of high-molecular-weight bands in which integrated genomes would appear and would be represented along with endogenous sequences bound to the probe. Therefore, if AAV2/5 did have a greater number of integrants the degree could not be determined. These confounding AAV5 observations have been made for the first time and require further investigation. Using AAV2/8 pseudotyped vectors in conjunction with the equal or greater expression potential of the liver-specific albumin promoter²⁶ may provide for the optimal combination for the down-regulation of AAT-PiZ. Through these data, we have made considerable progress toward developing a rAAV viral vector therapy capable of infecting a large enough population of cells within the liver, with the goal of down-regulating mutant transcripts in human AAT-deficiency patients by expressing therapeutic molecules.

Of importance when selecting a virus or serotype to use in gene therapy applications is the potential for integration and disruption of tumor suppressor and genes essential to the normal functioning and maintenance of the human body. In one retroviral gene therapy trial in particular, 2 of 10 treated patients acquired a leukemia condition resulting from the vector integration within the LMO2 oncogene and activation (reviewed in^27,28). This result was confirmed through sequencing of vector–chromosome junctions and the identification of clonal expansion of the disrupted genome. Ongoing clinical trials using AAV2-based vectors have shown them to be safe for up to 4 years²⁹. Chances of vector integration may be modeled in mouse studies with the use of Southern blotting. Our data presented here do not show the presence of migrating high-molecular-weight genomic DNA when total genomic DNA is digested with an enzyme that cuts once within the vector, indicating very low chances of integration. We cannot rule out the possibility of a very limited number of integrated genomes (<1 copy/cell) in the present study. Nevertheless, it will be critical to follow human patients through liver biopsy to address this issue fully.

Three major conclusions can be drawn from these data regarding the future of AAV as a gene vector for treating genetic disorders. First of all, the higher potency of AAV2/8 vectors investigated here can mean a safer application of the virus. When a lower dose of the virus is required to achieve the same therapeutic effect, the amount of antigen introduced and subsequent immunogenic potential of the vector can be reduced, as well as secondary site infections when introduced into the circulation. Secondary benefits of this attribute of AAV2/8 may be a safer drug and an increased potential for multiple injections without rejection if needed. Finally, given current production limits and costs accompanying the use of AAV at GLP and GMP standards for a clinical application, the ability to correct secreted and monogenic disorders with lower doses could considerably expand the utility of the system.

Methods

Producing and purifying rAAV vectors

The University of Florida Powell Gene Therapy Center produced rAAV serotype 1, 2, 5, and 8 vectors for these studies. The techniques for AAV2 used included heparan-affinity column chromatography as previously reported¹⁴. Virus production included use of the helper/packaging plasmid pDG, which supplies all the necessary helper functions as well as rep and cap in trans. Vector plasmid (622.5 g) pCB-AAT was cotransfected by calcium phosphate precipitation with 1867 g of pDG into one cell factory of 70–95% confluent 293 cells. Recombinant AAV was prepared by iodixanol centrifugation and hand-packed heparan column purification. Physical titer (genome number) was determined by dot blot. The infectious titer and extent of wild-type AAV2 contamination were determined by infectious center assay¹⁵. Spin concentrators were used to desalt. The packaging protocols for AAV pseudotypes are described in¹⁶. The AAV vectors produced were titered by dot blot assay and results in particles per milliliter were AAV2/1, 1.75 10¹³; AAV2/2, 6.16 10¹²; AAV2/5, 7.47 10¹³; and AAV2/8, 4.14 10¹².

Portal vein injection and partial hepatectomy

Eight-week-old female C57Bl/6 mice were purchased from the University of Florida Pathology Mouse Colony and handled as approved by the University of Florida Institutional Animal Care and Use Committee. Mice were randomly assigned to treatment group. For portal vein injections, all animals were anesthetized with 3% isoflurane. A ventral midline abdominal incision was made into the peritoneal cavity, and the portal vein was exposed. AAV vectors or PBS (100 l) were administered into the portal vein using a 30-gauge needle (BD Pharmagen). Hemostasis was achieved by application of a small piece of sterile swab directly onto the portal vein. Surgeries were performed on a thermoregulated operating board designed to maintain a temperature of 37°C and routinely lasted 10 min. For monitoring hAAT expression, serum samples were obtained via the tail vein at weekly intervals (0, 1, 2, 4, 6, 8, 12, 16, and 24).

At 24 weeks post-AAV administration, a partial hepatectomy was performed by excision of three lobes as previously described¹⁷. Although not adversely effecting the animal's normal behavior and organ system function, this procedure results in removal of up to 85% liver mass and initiates liver regeneration with nearly total liver regeneration by 4 weeks. Resected tissue was divided into samples for genomic DNA isolation and Southern/Q-PCR and histopathology.

Histopathology and immunohistochemistry

At 24 weeks post-AAV administration, mice were euthanized and blood samples harvested for serum. The liver and pancreas were completely removed. Representative samples were snap frozen for subsequent genomic DNA isolation or fixed in 10% neutral-buffered formalin for histology and immunohistochemistry.

Formalin-fixed paraffin-embedded tissue sections (4 m) were sequentially deparaffinized, rehydrated, and blocked for endogenous peroxidase activity. Following antigen retrieval in Target Retrieval solution (DakoCytomation, Carpinteria, CA, USA), slides were serum blocked and incubated with either rabbit anti-human AAT (1:100; Research Diagnostic Institute, Flanders, NJ, USA) or normal rabbit immunoglobulin as a negative control. Antibody binding was detected using the EnVision+ HRP kit (DakoCytomation) and DAB+ (DakoCytomation). Slides were counterstained using hematoxylin (Vector Labs, Burlingame, CA, USA) and mounted. Representative digital images were captured using a Zeiss Axioskop equipped with an Axiocam camera. Camera exposure settings were constant for all images.

Semiquantitative evaluation of human AAT immunoreactivity was performed by an independent observer blinded to treatment groups. Scoring was performed for six parameters (distribution of panlobular staining (diffuse versus regional), individual cell intensity (perivenous, periportal, pericentral), decile for numbers of intensely positive cells, and nuclear staining). Scores ranged from 0 to 3 (none, mild, moderate, intense).

ELISA for human 1-antitrypsin

Culture media and mouse serum levels of AAT were analyzed by ELISA as previously described with minor modifications¹⁸. Serum was diluted in PBS–0.5% Tween 20. The substrate was TMB peroxidase (KPL) and the stopping buffer used was H₃PO₄ (Fisher). Data are means standard deviation.

Genomic DNA isolation and Southern blot

Total liver genomic DNA was isolated from 300–400 mg of tissue by a previously published protocol¹⁸. Following digestion of tissue with proteinase K, restriction digests were performed with 40 g DNA with 100 units of either ApaI or HindIII (NEB) overnight at 37°C. The next day, 10 units of additional enzyme was added and incubated for 2 h. Genomic DNA (40 g/lane) was electrophoresed in a 1% agarose gel and transferred to membrane for subsequent Southern analysis as described¹⁸.

Genomic DNA extraction and quantitative PCR

Isolation of genomic DNA was performed with the Qiagen DNAeasy Tissue Kit and DNA concentrations (1:25) were determined. One microgram of DNA was used in all quantitative PCRs according to a previously used protocol in our lab¹⁹ and reaction conditions followed those recommended by the manufacturer to include 50 cycles of 94°C for 40 s, 37°C for 2 min, 55°C for 4 min, and 68°C for 30 s. Primer pairs were designed for the CMV enhancer/chicken -actin promoter as described²⁰ and a standard curve was established using the pCBAT plasmid.

References

1. Flotte, T. R., et al. (2004). Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults. Hum. Gene Ther. 15: 93–128. | Article | PubMed | ISI | ChemPort |
2. Perlmutter, D. H. (1993). Liver disease associated with alpha 1-antitrypsin deficiency. Prog. Liver Dis. 11: 139–165. | PubMed | ChemPort |
3. Brantly, M. L., et al. (1991). Use of a highly purified alpha 1-antitrypsin standard to establish ranges for the common normal and deficient alpha 1-antitrypsin phenotypes. Chest 100: 703–708. | PubMed | ChemPort |
4. Ogushi, F., et al. (1988). Evaluation of the S-type of alpha-1-antitrypsin as an in vivo and in vitro inhibitor of neutrophil elastase. Am. Rev. Respir. Dis. 137: 364–370. | PubMed | ChemPort |
5. Wewers, M. D., et al. (1987). Replacement therapy for alpha 1-antitrypsin deficiency associated with emphysema. N. Engl. J. Med. 316: 1055–1062. | PubMed | ChemPort |
6. Wewers, M. D., Casolaro, M. A. and Crystal, R. G. (1987). Comparison of alpha-1-antitrypsin levels and antineutrophil elastase capacity of blood and lung in a patient with the alpha-1-antitrypsin phenotype null(null before and during alpha-1-antitrypsin augmentation therapy. Am. Rev. Respir. Dis. 135: 539–543. | PubMed | ChemPort |
7. Casolaro, M. A., et al. (1987). Augmentation of lung antineutrophil elastase capacity with recombinant human alpha-1-antitrypsin. J. Appl. Physiol. 63: 2015–2023. | PubMed | ISI | ChemPort |
8. Levy, M. Y., Barron, L. G., Meyer, K. B. and Szoka, F. C. Jr. (1996). Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood. Gene Ther. 3: 201–211. | PubMed | ISI | ChemPort |
9. Qui, P., Zeigelhoffer, P., Sun, J. and Yang, N. S. (1996). Gene gun delivery of mRNA in situ results in efficient gene transgene expression and genetic immunization. Gene Ther. 3: 262–268. | PubMed | ISI | ChemPort |
10. Rosenfeld, M. A., et al. (1991). Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science 252: 431–434. | Article | PubMed | ISI | ChemPort |
11. Setoguchi, Y., Jaffe, H. A., Danel, C. and Crystal, R. G. (1994). Ex vivo and in vivo gene transfer to the skin using replication-deficient recombinant adenovirus vectors. J. Invest. Dermatol. 102: 415–421. | Article | PubMed | ISI | ChemPort |
12. Zern, M. A., et al. (1999). A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line. Gene Ther. 6: 114–120. | Article | PubMed | ChemPort |
13. Gao, G. P., et al. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 99: 11854–11859. | Article | PubMed | ChemPort |
14. Conway, J., et al. (1999). High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. Gene Ther. 6: 986–993. | Article | PubMed | ISI | ChemPort |
15. Walz, C. M., et al. (1998). Detection of infectious adeno-associated virus particles in human cervical biopsies. Virology 247: 97–105. | Article | PubMed | ChemPort |
16. Zolotukhin, S., et al. (2002). Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28: 158–167. | Article | PubMed | ISI | ChemPort |
17. Song, S., et al. (2004). DNA-dependent PK inhibits adeno-associated virus DNA integration. Proc. Natl. Acad. Sci. USA 101: 2112–2116. | Article | PubMed | ChemPort |
18. Song, S., et al. (2001). Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors. Gene Ther. 8: 1299–1306. | Article | PubMed | ChemPort |
19. Song, S., et al. (2002). Intramuscular administration of recombinant adeno-associated virus 2 -1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects. Mol. Ther. 6: 329–335. | Article | PubMed | ChemPort |
20. Donsante, A., et al. (2001). Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors. Gene Ther. 8: 1343–1346. | Article | PubMed | ISI | ChemPort |
21. Flotte, T., et al. (2001). Efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors. Diabetes 50: 515–520. | PubMed | ChemPort |
22. Zabner, J., et al. (2000). Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J. Virol. 74: 3852–3858. | Article | PubMed | ISI | ChemPort |
23. Qiu, J., Nayak, R., Tullis, G. E. and Pintel, D. J. (2002). Characterization of the transcription profile of adeno-associated virus type 5 reveals a number of unique features compared to previously characterized adeno-associated viruses. J. Virol. 76: 12435–12447. | Article | PubMed | ChemPort |
24. Song, S., Laipis, P. J., Berns, K. I. and Flotte, T. R. (2001). Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle. Proc. Natl. Acad. Sci. USA 98: 4084–4088. | Article | PubMed | ChemPort |
25. Thomas, C. E., Storm, T. A., Huang, Z. and Kay, M. A. (2004). Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. J. Virol. 78: 3110–3122. | Article | PubMed | ISI | ChemPort |
26. Xiao, W., et al. (1998). Adeno-associated virus as a vector for liver-directed gene therapy. J. Virol. 72: 10222–10226. | PubMed | ISI | ChemPort |
27. Kang, E. M. and Tisdale, J. F. (2004). The leukemogenic risk of integrating retroviral vectors in hematopoietic stem cell gene therapy applications. Curr. Hematol. Rep. 3: 274–281. | PubMed |
28. Relph, K., Harrington, K. and Pandha, H. (2004). Recent developments and current status of gene therapy using viral vectors in the United Kingdom. BMJ 329: 839–842. | Article | PubMed |
29. Manno, C. S., et al. (2003). AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101: 2963–2972. | Article | PubMed | ISI | ChemPort |

Acknowledgements

The authors acknowledge Tina Yanchis and Kristi Vale-Cruz, Molecular Pathology Core, for technical assistance. This work was supported by grants from the NIDDK (DK58327) and NHLBI (HL59412, HL69877) and the Alpha One Foundation.