Our group has developed two investigational clinical gene therapy products for gene augmentation of AAT as a potential therapy for the lung disease by using the recombinant adeno-associated viral vectors (rAAV2-AAT and rAAV1-AAT).^7,8 However, because a decrease in the expression of Pi*Z mutant protein is required to halt or ameliorate the hepatocellular damage, researchers tried to downregulate AAT messenger RNA (mRNA). One approach was to utilize hammerhead ribozymes designed to cleave AAT mRNA at a specific site.⁹ Another approach, is the application of RNA interference (RNAi) to decrease levels of the mutant mRNA transcript.¹⁰ In our lab, small short hairpin RNAs (shRNAs) were designed to downregulate endogenous AAT within hepatocytes.¹¹ Although this strategy proved effective, the optimal approach would be one that knocks down PiZ protein while at the same time increasing M-AAT protein. Our initial shRNA experiments relied on polymerase III U6-based promoters and did not show any liver-associated toxicity although we only followed mice for 4 weeks post shRNA delivery. However other groups have implicated AAV-mediated shRNA delivery with toxic side-effects, in one instance shRNA delivery to liver was associated with toxicity only when shRNAs greater that 19 base pairs in length.^12,13 In addition to controlling for the length of the shRNA it has also been shown that toxicity can be avoided employing RNAi with microRNAs (miRNAs) instead of shRNAs.¹⁴ Despite this, recently bicistronic AAV vectors expressing shRNAs against PiZ AAT and a codon-optimized AAT gene have been evaluated in mice. However the study had several limitations, mainly serum AAT protein detection showing effective knockdown of mutant and concomitant augmentation of normal AAT was not clearly demonstrated and was only convincingly shown by mRNA transcript analysis. In addition, these studies were limited to a duration of 2 weeks post AAV delivery and did not address long-term sustained knockdown and augmentation therapy or previous finding with shRNA-mediated liver toxicity.¹⁵

We have therefore examined a number of approaches for long-term expression of therapeutic miRNAs using the rAAV platform. miRNAs are single-stranded RNA molecules of about 21–23 nucleotides in length, that are able to regulate gene expression. These regulatory RNA molecules, which can be found in the intronic regions of protein coding genes of mammals, from stem loop structures that are recognized by a protein complex known as the microprocessor. This complex, consisting of the nuclease Drosha and the RNA-binding domain DGCR8, recognizes the pri-miRNAs stem loops and cleaves them out.¹⁶ The resulting pre-miRNAs are then exported out of the nucleus into the cytoplasm where they are processed by Dicer resulting in a mature 21–23 nucleotide miRNA that then enters the RNA-induced silencing complex thereby silencing its mRNA target.¹⁷ We have taken advantage of this pathway by altering the sequence of miR-155 to create artificial miRNAs that target the human AAT gene.

The optimization of this approach follows our previous study demonstrating efficient knockdown of mutant Z-AAT protein in PiZ-transgenic mice using a rAAV8 vector expressing U6-driven shRNAs.¹¹ Our in vivo experiments show a significant decrease in Z-AAT after administration of the rAAV9-intronCB-3xmiR-GFP vector (Figure 1c). Importantly these experiments highlight the additive effect that was obtained by using three anti-AAT miRNAs with different target sequences as none of the vectors with a single miRNA achieved the level of knockdown seen when they were delivered in combination (Figure 1c). The other biological effect aside from Z-AAT serum reduction that was observed included a significant and widespread decrease in the accumulation of Z-AAT within the hepatocytes and a reduction of the inflammatory lymphocyte foci within the liver (Figure 2). Endoplasmic reticulum accumulation of Z-AAT protein results in inflammation which overtime can lead to liver fibrosis and eventually to a failing cirrhotic liver.^4,5,6 Thus the reduction in inflammatory infiltrates as a result of the reduced burden of newly synthesized Z-AAT protein is important as it may halt the progression of liver disease and fibrotic tissue remodeling as a consequence of inflammation.

Further refinement of the anti-AAT miRNA efficacy was achieved by altering the location of the miRNA within the expression cassette. Initial short-term experiments demonstrated that expressing the miRNAs from the 3′ end of the GFP gene rather than from the intron of the CB promoter lead to a 25% increase in the silencing capabilities of the miRNAs and also to a significant decrease in the variability of this effect. Furthermore, doubling the effective miRNA dose per vector by having the miRNAs expressed from both locations led to more rapid onset of Z-AAT knockdown (Figure 4a). Importantly, increase in miRNA production was seen for both the PolyA-3XmiR-CB-GFP and the Double-6XmiR-CB-GFP vectors as compared to the rAAV9-intronCB-3xmiR-GFP vector. This seems to suggest that miRNA processing from the intron of the CB promoter is not as efficient as from the 3′ end of the GFP gene. However, the long-term experiments that followed showed that the initial kinetic differences in knockdown from the three vectors wanes overtime and by the 8 weeks the intronCB-3xmiR-GFP decreases in variability and augments in silencing efficacy. Western blot data from mice at day 90 analyzing the monomeric and polymeric Z-AAT fractions confirmed the effectiveness of miRNA-mediated knockdown seen in serum Z-AAT but also revealed more variability in the decrease of monomeric Z-AAT in mice receiving the intronCB-3xmiR-GFP vectors as compared to PolyA-3XmiR-CB-GFP and the Double-6XmiR-CB-GFP vectors (Figure 4b,c). More importantly this data clearly establishes that Z-AAT polymers are remarkably stable as there was no difference in the amount of Z-AAT polymers between GFP control mice and those receiving the anti-AAT miRNAs. Clearly, miRNAs would not be expected to decrease polymerized Z-AAT as they act on nascent Z-AAT mRNA; however, the remarkable stability of Z-AAT polymers throughout the 90-day study was surprising. The fact that there were no differences in the Z-AAT polymeric loads in the livers between groups of mice receiving GFP control vectors and anti-AAT miRNAs implies several things. First, this suggests that there was little new Z-AAT polymer formation during the 3-month study as one would expect that in the face of dramatic knockdown of newly synthesized Z-AAT as seen in the monomeric pool, few new polymeric aggregates should arise during the experiment as compared to control mice where newly synthesized Z-AAT monomers are not limiting. Second, the results also imply that the turnover of hepatocytes with Z-AAT polymers is rather slow, again as no differences were seen between the control and treated mice after 3 months. If there should have been a significant amount of hepatocytes turnover it would been expected that treated mice would have smaller Z-AAT polymer pools in the liver since these would not be as easily replenished in the presence of a Z-AAT knockdown.

The potency and stability of the decrease in serum and liver Z-AAT observed in vivo suggests that either of these vectors would lower Z-AAT levels in Pi*ZZ patients to therapeutic levels, even below those seen in Pi*MZ heterozygote patients. However maximal clinical benefit would be derived from a concomitant rise in M-AAT circulation. For this reason we designed the dual-function vectors to also deliver a miRNA-resistant M-AAT complementary DNA. The in vivo studies with these dual-function vectors clearly demonstrate the feasibility of concomitant knockdown and augmentation of mutant and wild-type protein respectively. These experiments also revealed that the double configuration of miRNAs had a more rapid onset of Z-AAT knockdown but the overall efficacy over time was comparable to the PolyA-3XmiR-CB-AAT vector. More importantly, the improved knockdown kinetics of the Double-6XmiR-CB-AAT vector came at a cost as was seen from the decreased output in M-AAT (Figure 5a). Initially, it was hypothesized that this may have been a result of decreased M-AAT mRNA production due to the presence of miRNA within the intron of this construct, but as shown in Figure 5c, there was not statistically significant difference in M-AAT mRNA between the two groups. It is possible that while mRNA transcription and stability are not affected by the presence of miRNAs within the intron, their translation into protein may be hindered as observed in the decrease circulating M-AAT levels in the serum of these mice.

If viral vector expressed miRNA-based therapy is to be translated into the clinic, one of the most pressing safety concerns is to determine what effect the expression of artificial miRNA has on endogenous miRNAs of the target organ? The question is a natural progression from the data that has been accumulating using first generation viral vector mediated RNAi. Emerging data from several labs using shRNA-mediated RNAi treatment for genetic disorders have reported severe adverse effects. An early landmark paper by Kay et al. described substantial hepatotoxicity and fatalities among mice receiving rAAV vector expressing shRNAs.¹² Similarly other early reports noted striatal neurotoxicity in mice treated with shRNA targeting the huntingtin gene and Purkinje cell loss in mice treated with shRNA against SCA1.^13,14,19 More recently these initial findings have been corroborated in a mouse model of the dominantly inherited neurological disease DYT1 dystonia. This study demonstrated striatal atrophy associated with behavioral dysfunction and death in mice receiving both therapeutic and control shRNA expressing rAAV vectors. Importantly the toxicity was encountered in wild-type and DYT1 mice.²⁰ Collectively these studies convincingly suggest that vectors expressing high amounts shRNAs driven by strong pol-III promoters (e.g., U6 and H1) have the potential to be highly toxic. Initial evidence from two studies implicated the exogenous shRNA constructs could cause dysfunction of the endogenous miRNA pathway by over-saturating key enzymes in the system.^12,21 A recent study extending this mechanism revealed that least in part adverse effect associated with shRNA expression are due to the saturation of several rate-limiting components in the miRNA pathway. Specifically it was noted that shRNA saturated exportin-5 which is involved in the nuclear export or pre-miRNAs as well as all four human Ago proteins.²² Saturation of Ago proteins has serious implications since these proteins are involved in miRNA biogensis, oogenesis, embryogensis, and a myriad of other cellular pathways^{23,24,25,26,27,28,29} To determine if our rAAV9 expressed anti-AAT miRNAs were disturbing the endogenous miRNA profiles of the liver, we interrogated the livers of 30 mice with a miRNA microarray. Neither did the delivery of rAAV9-GFP or those expressing miRNAs have a significant impact on miRNA profiles. Notably mir-122 which is the most abundant miRNA produced in the liver and regulates fatty acid metabolism was unaffected in any group. While a few miRNAs were found to be statistically different among the groups, they were mostly confined to a twofold change with the exception of miR-1. In the array experiments comparing untreated mice, miR-1 was one of the miRNAs that was found to be upregulated in PiZ mouse livers when compared to wild-type mice. More importantly downregulation to near wild-type levels of miR-1 was observed in all PiZ mice livers receiving rAAV9 (Figure 6a). Interestingly this correction of miR-1 levels was observed in the rAAV9-GFP group as well, suggesting that the downregualtion of miR-1 was not due to artificial miRNA expression by the vector but a consequence of rAAV9 transduction itself. While these results highlight the relatively unperturbed nature of miRNA pools in the liver after artificial miRNA delivery with rAAV9, it does not address whether endogenous miRNA function is affected. Recent studies by Khan et al. demonstrate that shRNA, miRNAs, and small interfering RNAs (siRNAs) transfection in vitro can all lead to altered gene regulation as RNA-induced silencing complex or machinery downstream of exportin-5 is saturated.³⁰ Their findings included the de-repression of miRNA-regulated genes as transfected small RNAs presumably competed with endogenous miRNAs for loading onto the RNA-induced silencing complex. These findings warrant further research using gene micorarrays to assess the consequences of rAAV delivered miRNAs on global gene profiles in vivo. Thus in summary, miRNA profiles seemed unperturbed and in some cases “corrected ” back to wild-type levels with rAAV9 delivery.

Cell culture and transfection. HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 mg/l of penicillin-streptomycin (Gemini Bio-products, West Sacramento, CA). Cells were maintained in a humidified incubator at 37 °C and 5% CO₂. Plasmids were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Cell culture supernatants were collected at 24, 48, and 72 hours, and cell lysates were collected at 72 hours.

Serum AAT ELISAs. Human AAT ELISA: Total AAT protein was detected by ELISA. High binding extra, 96-well plate (Immulon 4; Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of human specific goat anti-AAT (1:500 diluted; Biomedicals, Solon, OH) in Voller's buffer overnight at 4 °C. After blocking with 1% non-fat dry milk in phosphate-buffered saline with Tween 20, duplicate standard curves (Athens Research and Technology, Athens, GA) and serially diluted unknown samples were incubated in the plate at room temperature for 1 hour, a second antibody, goat anti-hAAT(HRP) (1:5,000 diluted; Abcam, Cambridge, MA) was incubated at room temperature for 1 hour. The plate was washed with phosphate-buffered saline-Tween 20 between reactions. After reaction with TMB peroxidase substrate (KPL, Gaithersburg, MD) reactions were stopped by adding 2 N H₂SO₄ (Fisher Scientific, Hudson, NH). Plates were read at 450 nm on a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA).

Z-AAT ELISA: Human Z-AAT protein levels were detected by ELISA with coating antibody specific for human Z-AAT (1:100 diluted mouse-anti-human α-1-antitrypsin-Z; Cell Sciences, Fair Lawn, NJ). Standard curves were created with PiZ mouse serum with 5% bovine serum albumin (Sigma, St Louis, MO). Serially diluted unknown samples were incubated in the plate at 37 °C for 1 hour. The secondary antibody and the next step were same as the standard human-AAT ELISA described above, except the secondary antibody was diluted in 5% bovine serum albumin and incubated in the plate at 37 °C for 1 hour.

c-Myc ELISA: c-Myc tag levels were quantified by a method similar to that described above. Plates were coated with a c-Myc antibody (1:1,000 diluted Goat anti-c-Myc; Abcam), plates were then blocked with 5% bovine serum albumin at 37 °C for 1 hour. To create the standard curve C57Bl/6 mice were dosed via tail vein with c-Myc-AAT expressing vector, at 2 weeks serum were collected and pooled. The amount c-Myc-AAT in the serums was then quantified with the human specific AAT ELISA described above. The obtained values were then used to produce a standard of c-Myc protein for the c-Myc ELISA.

RNA extraction: Flash frozen mouse liver tissue was ground in a pestle and mortar and used to extract either small or total RNA using the mirVana miRNA RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions.

microRNA qRT-PCR: mircoRNA was primed and reverse-transcribed with TagMan MicroRNA reverse transcription Kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed in duplicate with gene-specific RT-miRNA primers, and PCR assays were as designed by Applied Biosystems, using TaqMan Gene Expression Master mix (Applied Biosystems) in a StepOne Plus real-time PCR instrument (Applied Biosystems).

PiM and PiZ qRT-PCR: Total RNA was primed with oligo(dT) and reverse-transcribed with SuperScript III First-Strand Synthesis kit for RT-PCR (Invitrogen). Quantitative PCR were performed by gene-specific primer pairs. PiM and PiZ share the primers but differ in the probes. Forward primer CCAAGGCCGTGCATAAGG, reverse primer: GGCCCCAGCAGC TTCAGT, PiZ probe: 6FAM-CTGACCATCGACAAGA-MGBNFQ, and PiM probe: 6FAM-CTGACCATCGACGAGA-MGBNFQ. Reactions were performed using TaqMan Gene Expression Master mix (Applied Biosystems) in a StepOne Plus real-time PCR instrument (Applied Biosystems).

Z-AAT transgenic mice and rAAV9 delivery. The PiZ-transgenic mice used in this study have been described.¹¹ All animal procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. rAAV9 vector was administered by mouse tail vein injection in a volume of 200 µl with titer of 1 × 10¹² vps. The injections were performed in the most accessible vessels veins that run the length of both lateral aspects of the tail by grasping the tail at the distal end. Blood was collected through the facial vein pre-injection and every week after tail vein rAAV9 delivery until termination of the studies.

Liver histology. For determination of histological changes, liver samples were fixed in 10% neutral-buffered formalin (Fisher Scientific), and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin and PAS with or without diastase digestion.

Immunohistochemistry for hAAT, was performed as described,¹³ briefly tissue sections (5 µm) were deparaffinized, rehydrated, and blocked for endogenous peroxidase with 3% hydrogen peroxide in methanol for 10 minutes. To detect hAAT expression, tissue sections were incubated with primary antibody, rabbit antihuman AAT (1:800; RDI/Fitzgerald Industries, Acton, MA), for overnight at 4 °C. Staining was detected using ABC-Rb-HRP and DAB kits (Vector Laboratories, Burlingame, CA).

Histology image analysis. Slides were stained for PASD to remove glycogen. Whole digital slide images were created using an Aperio CS ScanScope (V, CA) and analyzed using the positive pixel count algorithm (version 9). PASD-positive globules were expressed as the proportion of strong positive pixels to total pixels using a hue value of 0.9, hue width of 0.15, and color saturation threshold of 0.25. The intensity threshold for strong positivity was set to an upper limit of 100.

Analysis of Z-AAT protein monomer and polymer. For soluble/insoluble protein separation, 10 mg of whole liver was added to 2 ml buffer at 4 °C (50 mmol/l Tris-HCl (pH 8.0), 150 mmol/l NaCl, 5 mmol/l KCl, 5 mmol/l MgCl₂, 0.5% Triton X-100, and 80 µl of complete protease inhibitor stock). The tissue was homogenized in a prechilled Dounce homogenizer for 30 repetitions, then vortexed vigorously. A 1-ml aliquot was passed through a 28-gauge needle 10 times. The total protein concentration of the sample was determined, and a 5-µg total liver protein sample was aliquoted and centrifuged at 10,000g for 30 minutes at 4 °C. Supernatant (soluble (S) fraction) was immediately removed into fresh tubes; extreme care was taken to avoid disturbing the pellet (insoluble (I) fraction). The insoluble polymers pellet (I fraction) was denatured and solubilized via addition of 10 l chilled cell lysis buffer (1% Triton X-100, 0.05% deoxycholate, 10 mmol/l EDTA in phosphate-buffered saline), vortexed for 30 seconds, sonicated on ice for 10 minutes and vortexed. To each soluble and insoluble sample, 2.5 sample buffer (50% 5 sample buffer (5% sodium dodecyl sulfate, 50% glycerol, 0.5 mol/l Tris (pH 6.8)), 10% mercaptoethanol, 40% ddH₂O) was added at a volume of 50% of the sample volume. Samples were boiled and loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); equal amounts of total liver protein were loaded per soluble–insoluble pair in quantitative experiments. Densitometry was performed using Image J Software (NIH, Bethesda, MD).

Serum chemistry. All serum samples were analyzed by UMass Mouse Phenotyping Center Analytical Core, using the NExCT Clinical Chemistry Analyzer (Alfa Wassermann Diagnostic Technologies, West Caldwell, NJ). Serum was analyzed for alanine aminotransferase and aspartate aminotransferase according the manufacturers specifications.

miRNA microarray expression analysis. We isolated 8 µg of total RNA from flash frozen mouse livers using the mirVana miRNA isolation kit (Ambion). The experimental design included six groups with RNA samples from five mice each which were assayed on single color arrays for a total of 30 independent microarrays. In brief, the RNA was labeled with Cy5 and hybridized to dual-channel microarray µParaFlo microfluidics chips (LC Sciences, Houston, TX) containing miRNA probes to mouse mature miRNAs available in the Sanger miRBase database (Release 16.0) as described.³² Each of the spotted detection probes consisted of a nucleotide sequence complementary to a specific miRNA sequence and a long non-nucleotide spacer that extended the specific sequence away from the chip surface. Fluorescence images were collected with a laser scanner (GenePix 4000B; Molecular Devices) and digitized using Array-Pro image analysis software (Media Cybernetics, Bethesda, MD). The data were analyzed, including background subtraction, with a LOWESS (locally weighted regression) method on the background-subtracted data as described.³³ The normalization is to remove system related variations, such as sample amount variations and signal gain differences of scanners. Samples were considered positive only if transcripts had a signal intensity higher than 3 times (background-subtracted data) and the spot coefficient of variation <0.5. Coefficient of variation as calculated by (subtracted data)/(signal intensity), and in which repeating probes on the array produced signals from at least 50% of the repeating probes above detection level. Data are represented as a Log₂ transformation. The data were further filtered to remove miRNAs with (normalized) intensity values below a threshold value of 32 across all samples. t-test were performed between “control ” and “test ” sample groups where T values are calculated for each miRNA, and P values are computed from the theoretical t-distribution. If P ≤ 0.05, it is plotted as red spot in a log scatter plot.

Figure S1.

Figure S2.

Figure S3.