Deposition and Expression of Aerosolized rAAV Vectors in the Lungs of Rhesus Macaques
Authors: Suzanne E. Beck, Beth L. Laube, Carolina I. Barberena, Anne C. Fischer, Robert J. Adams, Kye Chesnut, Terence R. Flotte, William B. Guggino
Journal: Molecular Therapy (2002)

The goals of these experiments were to efficiently deliver aerosolized adeno-associated virus (AAV) vector to the lungs of Rhesus macaques and to measure gene transfer and expression. To determine optimal lung deposition, we compared four techniques of delivering aerosolized saline admixed with the radioisotope 99mtechnetium (99mTc) nebulized through a mouthpiece (Neb Oral), a laryngeal airway mask (Neb LMA), or an endotracheal tube (Neb ETT), or bronchoscopically delivered by Microsprayer (PennCentury). Total lung deposition fraction, as indicated by gamma scintigraphy, averaged 0.5% (Neb Oral), 1.2% (Neb LMA), 1.8±0.4% (Neb ETT), and 62.3±11.3% (Microsprayer). Because microspraying was the most efficient method of delivery, we used it to administer saline with 99mTc-labeled diethylene-triamine penta-acetic acid (DTPA) admixed with 9×1011 infectious units (i.u.) of AAV serotype 2 (rAAV2) vector encoding green fluorescent protein (GFP; rAAV2-GFP). Initial total and regional lung depositions were quantified by scintigraphy. We analyzed the tissue three weeks later for vector-specific DNA transduction and RNA expression. Radioisotope was detected in all lung regions, reflecting an average dose of 1.33×1010±9.5×109 i.u. per region. Regional data indicated an increase in expression when the dose exceeded 3×109 i.u. (P=0.030). We conclude that expression of rAAV2-GFP in lungs appears to be related to depositing a regional threshold dose greater than 3×109 i.u., easily achieved by bronchoscopic microspraying. The goals of these experiments were to efficiently deliver aerosolized adeno-associated virus (AAV) vector to the lungs of Rhesus macaques and to measure gene transfer and expression. To determine optimal lung deposition, we compared four techniques of delivering aerosolized saline admixed with the radioisotope 99mtechnetium (99mTc) nebulized through a mouthpiece (Neb Oral), a laryngeal airway mask (Neb LMA), or an endotracheal tube (Neb ETT), or bronchoscopically delivered by Microsprayer (PennCentury). Total lung deposition fraction, as indicated by gamma scintigraphy, averaged 0.5% (Neb Oral), 1.2% (Neb LMA), 1.8±0.4% (Neb ETT), and 62.3±11.3% (Microsprayer). Because microspraying was the most efficient method of delivery, we used it to administer saline with 99mTc-labeled diethylene-triamine penta-acetic acid (DTPA) admixed with 9×1011 infectious units (i.u.) of AAV serotype 2 (rAAV2) vector encoding green fluorescent protein (GFP; rAAV2-GFP). Initial total and regional lung depositions were quantified by scintigraphy. We analyzed the tissue three weeks later for vector-specific DNA transduction and RNA expression. Radioisotope was detected in all lung regions, reflecting an average dose of 1.33×1010±9.5×109 i.u. per region. Regional data indicated an increase in expression when the dose exceeded 3×109 i.u. (P=0.030). We conclude that expression of rAAV2-GFP in lungs appears to be related to depositing a regional threshold dose greater than 3×109 i.u., easily achieved by bronchoscopic microspraying. IntroductionThe goal of gene therapy in cystic fibrosis (CF) lung disease is to overcome native cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction by placing normal copies of CFTR cDNA in airway cells to achieve normal protein expression. In humans, the potential targets for this therapy appear to be the epithelial cells of large and small airways, and the submucosal glands, where CFTR protein is expressed [1Engelhardt J. Submucosal glands are the predominant site of CFTR expression in the human bronchus.Nat. Genet. 1992; 2: 240-248Crossref PubMed Scopus (566) Google Scholar].Adeno-associated virus type 2 (AAV2) vectors are promising candidates to deliver a normal copy of CFTR to the respiratory tract of patients with CF. In early studies in New Zealand White (NZW) rabbit and Rhesus macaque in vivo models, recombinant adeno-associated virus (rAAV)-CFTR vectors, delivered directly to the endobronchial surface by local bronchoscopic instillation of doses up to 109 DNase-resistant particles, yielded localized AAV-mediated DNA transfer and protein expression in the airway epithelium [2Flotte T. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.Proc. Natl. Acad. Sci. USA. 1993; 90: 10613-10617Crossref PubMed Scopus (442) Google Scholar, 3Conrad C. Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung.Gene Ther. 1996; 3: 658-668PubMed Google Scholar, 4Beck S.E. Repeated delivery of adeno-associated virus vectors to the rabbit airway.J. Virol. 1999; 73: 9446-9455Crossref PubMed Google Scholar]. These initial studies encouraged the development of clinical trials using rAAV-CFTR in patients with CF. Two clinical studies investigated AAV-CFTR delivery directly to the sinus epithelia of adults with CF and demonstrated gene transduction up to 10 weeks after initial instillation [5Wagner J. A phase I/II study of tgAAV-CF for the treatment of chronic sinusitis in patients with cystic fibrosis.Hum. Gene Ther. 1998; 9: 889-909Crossref PubMed Scopus (91) Google Scholar, 6Wagner J. Efficient and persistent gene transfer of AAV-CFTR in maxillary sinus.Lancet. 1998 Bb; 351: 1702-1703Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar].Although these results are promising, a number of hurdles must be overcome before pulmonary rAAV gene therapy is realized as an effective treatment. First, there are a limited number of AAV receptors in the apical membrane [7Zabner J. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitate gene transfer.J. Virol. 2000; 74: 3852-3858Crossref PubMed Scopus (269) Google Scholar, 8Bals R. Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.J. Virol. 1999; 73: 6085-6088PubMed Google Scholar, 9Duan D. Yue Z.Yan, P. McCray J. Engelhardt J. Polarity influences the efficiency of recombinant adeno-associated virus infection in differentiated airway epithelia.Hum. Gene Ther. 1998; 9: 2761-2776Crossref PubMed Scopus (155) Google Scholar, 10Teramoto S. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.J. Virol. 1998; 72: 8904-8912Crossref PubMed Google Scholar], which may limit the ability of the virus to enter the cell following airway delivery. Second, vector entry into cells by way of the apical membrane results in altered endosomal processing and nuclear trafficking [11Duan D. Yue Y. Yan Z. Yang J. Engelhardt J. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus.J. Clin. Invest. 2000; 105: 1573-1587Crossref PubMed Scopus (304) Google Scholar] when compared with basolateral entry, possibly limiting effective gene transduction. Third, rAAV infection and transduction appear to be inhibited in vitro by excessive local proinflammatory mediators and neutrophil elastase, which are present in the bronchoalveolar lavage (BAL) fluid of CF patients [12Virella-Lowell I. Poirier A. Chesnut K. Brantly M. Flotte T. Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients.Gene Ther. 2000; 7: 1783-1789Crossref PubMed Scopus (79) Google Scholar]. In an attempt to overcome these barriers, higher titer vectors are being produced, and innovative strategies, such as altering endocytic processing [13Hansen J. Qing K. Srivastava A. Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts.J. Virol. 2001; 75: 4080-4090Crossref PubMed Scopus (121) Google Scholar], alternative receptor targeting, and reducing preexisting inflammation, are being developed.Given the potential barriers of rAAV-mediated gene transduction in airway epithelial cells, delivery of vector and deposition in the lung must be optimized. Thus, the aim of this study was to determine an efficient mode for delivering aerosolized rAAV vector to the lower airways of Rhesus macaques and to measure subsequent DNA transfer and expression of a reporter gene.ResultsDetermining an Efficient Mode of Aerosol DeliveryIn these experiments, we compared various modes for delivering nebulized saline particles to bronchoscopic microspraying.Nebulization at the mouth (Neb Oral) of 99mTc-DTPA-saline (saline with 99mTc-labeled diethylene-triamine penta-acetic acid (DTPA)) aerosol resulted in a total pulmonary deposition fraction of 0.5%. Although the distribution in the lung was uniform, most deposition occurred on the face or in the oral cavity (Fig. 1A).Nebulization through the laryngeal airway mask (LMA; Neb LMA, data not shown) and endotracheal tube (ETT; Neb ETT, Fig. 1B) avoided the high losses in the oral cavity observed with Neb Oral delivery, but directed aerosol to the glottis or midtrachea, respectively. Aerosol that penetrated to the lower airways with these two modes of delivery deposited uniformly. However, the total pulmonary deposition fraction was only 1.2% and 1.8 ± 0.4%, respectively.99mTc-DTPA-saline aerosol delivered bronchoscopically by the MicroSprayer device (PennCentury, Philadelphia, PA) resulted in an enhanced pulmonary deposition fraction compared with delivery by nebulization. Total deposition fraction averaged 62.3 ± 11.3% (Fig. 1D), with similar amounts of aerosol deposited in both lungs.Bronchoscopic instillation of 99mTc-DTPA-saline through an ETT resulted in the highest total deposition fraction, averaging 91.8 ± 11.3% (Fig. 1C). However, the pattern of deposition was inconsistent among the three animals, with deposition favoring the right lung in one monkey and the left lung in another.The most efficient method of delivery was bronchoscopic delivery with the MicroSprayer, which resulted in a 50- to 100-fold enhancement in aerosol deposition compared with nebulization modes and in a uniform distribution between the right and left lungs.Deposition of rAAV2-GFP Vector Aerosol in the Lungs of Three MacaquesDeposition of aerosol containing 99mTc-DTPA-saline admixed with 9 × 1011 infectious units (i.u.) of AAV serotype 2 (rAAV2) vector encoding green fluorescent protein (GFP), AAV2-GFP was quantified from the initial gamma camera image of the anterior lung following bronchoscopic delivery with the MicroSprayer device. Deposition of radioisotope in the right lung averaged 50.1 ± 9.0% of the starting dose. Deposition in the left lung averaged 30.3 ± 15.5%. This meant that, on average, a total of 4.5 ± 0.8 × 1011 i.u. and 2.73 ± 1.40 × 1011 i.u of vector deposited in the right and left lungs, respectively.Deposition favored the central portion of the right lung, with little if any aerosol penetrating to the lung periphery (Fig. 1D). However, this visual appearance was due to an instrumental masking of regions with subthreshold count values. When deposition in each of nine regions was quantified (Fig. 2B), the masking effect was eliminated. With quantification, we determined that the aerosol deposited not only in the central regions of the lungs, but also in the peripheral lung regions. Average deposition, expressed as a percent of total right lung deposition, was 0.23 ± 0.25%, 3.76 ± 5.53%, 3.15 ± 2.94%, 3.02 ± 1.39%, 14.32 ± 2.76%, 12.50 ± 3.23%, 2.22 ± 0.61%, 6.96 ± 1.66%, and 3.95 ± 0.35% in regions 1–9, respectively. Multiple comparisons with a Kruskal–Wallis test indicated that deposition was similar in regions 2–9, whereas deposition in region 1 was statistically significantly different from that in regions 5 and 6.FIG. 2Regional deposition. A nine-region grid was delineated on the 133Xe gas image to determine functional lung borders (A) and superimposed on the 99mTc aerosol image (B) of each macaque. The lungs of each macaque were removed and divided into a nine-region grid (C) that was identical to the one created for the initial aerosol image 3 weeks after the administration of 99mTc-rAAV2-GFP by bronchoscopic microspraying.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Obtaining the weight of the right lung regions at necropsy, we further expressed deposition as dose (i.u.)/g of tissue in each region to normalize regional deposition by the differences in regional mass. Normalizing regional deposition resulted in less variation between the regions, and there were no statistically significant differences (Fig. 3A).FIG. 3Average dose and gene transfer in each right lung region. (A) Average dose (i.u.) of 99mTc-rAAV2-GFP admixture per g of tissue in each right lung region (n = 3 for each region). Variability between regions was not statistically significant. (B) Average copy number of rAAV2-GFP-DNA per g of tissue in each right lung region (n = 3 for each region). Variability between regions was not statistically significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)rAAV2-Mediated GFP cDNA Transfer in LungsrAAV2-GFP DNA, detected in each lung region, was also normalized in terms of regional mass and expressed in terms of copy number/g of right lung tissue (Fig. 3B). GFP DNA was detected in every region, and differences between regions were not statistically significant. No GFP DNA was detected in the control animal.Relationship Between Regional Dose and Regional DNA TransferA Spearman rank-correlation test indicated that regional DNA transfer, expressed as copy number/g of tissue in each region, was not statistically significantly associated with regional dose, expressed as i.u./g of dose between regions.rAAV2-Mediated GFP cDNA Transfer in Nontargeted TissuerAAV2-GFP DNA transfer in nontargeted tissue is shown in Table 1 for each macaque. Vector-specific DNA was detected in adjacent tissue, such as the trachea and oropharynx, at 1% of the levels found in the targeted lung and oropharynx. Vector-specific DNA was not detected in the control animal or in any immunologically privileged site, such as the brain and the reproductive organs, within the experimental group (Table 1).TABLE 1rAAV2-GFP DNA transfer in nontargeted tissueTissue12J19J22JThymus−−−Heart−−−Trachea1.89 × 1059.77 × 1022.61 × 102Tracheal lymph nodesatracheal−−n/aPharynx−−−Tongue−4.31 × 1012.59 × 100Visceral organsbVisceral organs included kidney, liver, and pancreas.−3.68 × 101−GI tractcGI tract included stomach, duodenum, jejunum, and colon. N/A, not sampled. GFP DNA was quantified by real-time PCR. Values represent copies/g of tissue. (−), no DNA detected.−1.71 × 104−Spleen−−−Brain−−−Muscle−−−Gonads−−−Bronchial lymph nodesdbronchial−N/A−Mesenteric lymph nodesemesenteric−−−Pharyngeal lymph nodesfpharyngeal lymph nodes (LN). The identification numbers of each monkey are 12J, 19J, and 22J.N/AN/A−DNA transfer was measured from the first round of nested PCR of samples taken froma trachealb Visceral organs included kidney, liver, and pancreas.c GI tract included stomach, duodenum, jejunum, and colon. N/A, not sampled. GFP DNA was quantified by real-time PCR. Values represent copies/g of tissue. (−), no DNA detected.d bronchiale mesentericf pharyngeal lymph nodes (LN). The identification numbers of each monkey are 12J, 19J, and 22J. Open table in a new tab rAAV2-Mediated GFP RNA ExpressionTable 2 shows the regional vector dose, regional DNA, and presence of RNA expression. Of the 27 regions (9 regions in each of three monkeys), 20 of 22 regions (91%) indicated expression when the dose exceeded 3 × 109 i.u. Assuming the 27 regions were independent samples, a Fisher exact test indicated that doses exceeding 3 × 109 i.u. resulted in a statistically significant increase in tissue specimens with vector RNA expression (P = 0.030; Fig. 4A). Figure 4B demonstrates the products of RT-PCR confirming RNA expression.TABLE 2Regional dose, regional rAAV2-GFP-DNA transfer, and RNA expression12J19J22JDose (i.u./g)Copy no./g tissueRNA expression 2nd round RTDose (i.u./g)Copy no./g tissueRNA expression 2nd round RTDose (i.u./g)Copy no./g tissueRNA expression 2nd round RT6.57 × 1084.88 × 101pos4.59 × 1083.23 × 103neg3.40 × 1092.24 × 105pos8.63 × 1083.91 × 102neg2.75 × 1091.87 × 103pos2.88 × 10101.59 × 106pos2.65 × 1096.00 × 103neg6.43 × 1091.63 × 105pos3.29 × 10101.91 × 105neg7.86 × 1096.12 × 103pos1.18 × 10103.74 × 103pos5.39 × 1099.16 × 104pos2.37 × 10101.33 × 105pos2.33 × 10107.05 × 104pos1.80 × 10102.11 × 104pos2.01 × 10101.53 × 104pos2.24 × 10105.78 × 104pos1.23 × 10105.48 × 106pos7.77 × 1091.82 × 102pos1.64 × 10106.18 × 103pos1.87 × 10101.18 × 105pos1.28 × 10102.69 × 104pos3.01 × 10101.11 × 103neg1.80 × 10107.98 × 106pos1.02 × 10103.23 × 104pos1.47 × 10101.08 × 103pos7.41 × 1098.61 × 104posDose (i.u./g tissue), copy number/g tissue, and positive (pos) or negative (neg) RNA expression in the second round of nested RT-PCR are shown for each region of the right lung. The identification numbers of each monkey are 12J, 19J, and 22J. Open table in a new tab FIG. 4rAAV2-GFP dose, transduction, and RNA expression. (A) Relationship between transduction and dose. Regional transduction was measured and plotted against the calculated regional dose for each specimen (n = 27). Vector-specific RNA expression is indicated as present (black squares) or absent (white squares). Regional calculated doses that exceeded 3 × 109 i.u. (vertical line) resulted in a statistically significant increase in vector expression (P = 0.030). (B) rAAV2-GFP RNA expression. The top panel shows the RT+ reaction for right-lung samples in regions 5–9 (lanes 1–5). Positive expression was found in lung regions 6, 7, 8, and 9 (lanes 2–5, respectively). The RT– reaction (bottom panel) verifies no contamination of DNA in the reaction as well as the GFP plasmid controls at 10−12 g, 10−15 g, and 10−18 g (lanes 6–8, respectively). Lane M is the marker and lane 9 is the water blank.View Large Image Figure ViewerDownload Hi-res image Download (PPT)rAAV2-Mediated GFP Protein ExpressionGFP expression was detected in the epithelial cells of the small and large airways (Figs. 5A and 5B). GFP expression was seen in the cytoplasm of ciliated columnar epithelial cells of the proximal airways (Fig. 5B) at the site of delivery. There was no evidence of acute or chronic inflammation in the airways, interstitium, or alveolar spaces. There was no expression detected in the epithelia or submucosal glands of the control animal (Fig. 5C, right). rAAV2-mediated GFP protein expression was also verified by western blot analysis (Fig. 5D).FIG. 5rAAV-mediated GFP protein expression. Sections of formalin-fixed lung, stained with hematoxylin and eosin, show (A) normal alveolar architecture (left), (B) intact ciliated epithelia and airway architecture (left), and (C) minimal lymphocytic infiltration (left). Right panels show sections analyzed for GFP-specific fluorescence by microscopy. GFP expression is evident in the epithelial cells of the (A) central (right) and (B) distal airways (right). Arrows indicate fluorescent airway epithelial cells. (C) The control animal did not demonstrate GFP fluorescence (right). Original magnification, ×200. (D) Western blot analysis of GFP (top) and β-actin (bottom) is shown. Lanes 1 and 2 contain GFP protein (positive control). Lanes 3–6 contain samples from different lung regions of the control animal. Lanes 7 and 8 demonstrate GFP protein present in treated animals. Of the samples tested from treated animals, two of six were positive for GFP expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionOne of the challenges in pulmonary gene therapy for CF is the efficient delivery of normal copies of CFTR to target cells. In our first set of experiments, we compared the efficiency of delivering this genetic material as an aerosol in a Rhesus macaque model. Nebulization was chosen as one delivery modality because it is convenient and established clinically for aerosol delivery. Microspraying was chosen because previous results indicated this modality led to the delivery of high doses of vector in human airway cast-models [14Cipolla D. Coarse spray delivery to a localized region of the pulmonary airways for gene therapy.Hum. Gene Ther. 2000; 11: 361-371Crossref PubMed Scopus (24) Google Scholar]. In our experiments, radiolabeled saline aerosol served as proxy for aerosolized vector. Nebulization to the mouth (Neb Oral) resulted in deposition that was uniform but very inefficient. Total lung deposition was only 0.5% of the initial dose, and the majority was deposited in the oropharynx or on the face of the animal. This degree of deposition is consistent with another study that delivered nebulized radiolabeled aerosol to macaques [15Pillai R. Hughes B. Wolff R. Heisserman J. Dorato M. The effect of pulmonary delivered insulin on blood glucose levels using two nebulizer systems.J. Aerosol. Med. 1996; 9: 227-240Crossref PubMed Scopus (11) Google Scholar]. Other reports of nebulized aerosol delivery showed that deposition ranged from 2 to 32% of the starting dose in adults [16Ruffin R. Obminski G. Newhouse M. Aerosol salbutamol administration by IPPB: lowest effective dose.Thorax. 1978; 33: 689-693Crossref PubMed Scopus (24) Google Scholar, 17Wasnich R. A high frequency ultrasonic nebulizer system for radioaerosol delivery.J. Nuc. Med. 1976; 17: 707-710PubMed Google Scholar, 18Lin M. Hayes T. Goodwin D. Kruse S. Distal penetration in a radioaerosol inhalation with an ultrasonic nebulizer.Radiology. 1974; 112: 443-447Crossref PubMed Scopus (12) Google Scholar] and from 0.2 to 2% in infants with CF [19Chua H. The influence of age on aerosol deposition in children with cystic fibrosis.Eur. Respir. J. 1994; 7: 2185-2191Crossref PubMed Scopus (174) Google Scholar].It is likely that such low deposition percentages will be insufficient for successful CFTR gene transfer and will be economically prohibitive. Therefore, we attempted to improve total pulmonary deposition by overcoming the loss of aerosol on the face and oropharynx. For those experiments, we tested nebulized delivery through artificial airway devices (Neb LMA, Neb ETT) and found that pulmonary deposition improved to approximately 2% of the initial dose. However, 98% of our initial dose still remained in the delivery system or was lost to the epiglottis or trachea. Direct instillation of 99mTc-DTPA-saline significantly enhanced pulmonary deposition to 91% as compared with nebulization, but led to uneven deposition patterns.Because of the high losses associated with nebulized delivery and the inconsistent deposition pattern associated with instillation, bronchoscopic microspraying was investigated. Microspraying is more invasive, requiring anesthesia and instrumentation of the airway, but it allows for delivery of high doses of rAAV vector to the airway surface. Another group of investigators has used a similar technology to deliver adenovirus vector to the lungs of normal individuals, demonstrating transient gene expression [20Harvey, B.-G., Hackett, N. R., Ely, S., and Crystal, R. G.2001. Host responses and persistence of vector genome following intrabronchial administration of an E1–E3– adenovirus gene transfer vector to normal individuals. Mol. Ther.3: 206–215,Google Scholar]. They did not quantify deposition patterns or deposition fraction in those experiments, whereas we have analyzed the deposition patterns from nebulization or direct instillation of vector.The data indicated that bronchoscopic microspraying was more efficient than nebulizer/compressor systems in delivering CFTR vectors to the lungs of macaques, and deposition was more evenly divided between the two lungs compared with deposition by direct instillation.It also appears that the dose of vector was distributed equally to both the large, central airways (represented by regions 5 and 6 in our nine-region grid) and to the small airways (represented by regions 1–4 and 7–9), when normalized for tissue weight. It is understood that the radioactivity detected in each of the nine regions also reflected alveolar activity. Nevertheless, based on the anatomy of the lung, we assumed regions 5 and 6 were composed predominantly of large, central airways, whereas the other regions were composed predominantly of small airways and alveoli.Results from our second group of experiments indicated that the efficient transfer of genetic material to large and small airway cells is feasible by microspraying a vector containing the genetic material, as vector-specific DNA was detected in every region of the right lung sections.Aitken and colleagues [21Aitken M.L. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease.Hum. Gene Ther. 2001; 12: 1907-1916Crossref PubMed Scopus (198) Google Scholar] recently delivered an adeno-associated virus encoding the human CFTR cDNA, tgAAVCF, as an aerosol to the lungs of patients with CF using the same Pari LC Plus nebulizer/ProNeb compressor system that we used in this study. Although 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of a dose of 1013 particles, RNA-specific PCR did not detect vector-derived mRNA at any time point in Aitken's study. This is in contrast to our study, in which a similar dose (that is, 1011 i.u.) resulted in gene expression 30 days after administration by microspraying. Deposition fraction was not quantified in Aitken's study, and our data suggest that it was probably significantly less than with microspraying. In addition, results from our study in Rhesus macaques indicate that gene expression may be related to depositing a regional threshold dose that is greater than 3 × 109 i.u. It is likely that exceeding a regional threshold dose is also necessary in humans, and achieving that dose will require technologies more efficient than nebulization, such as microspraying.In these experiments, we assumed that during the few minutes that were needed to administer the aerosol and acquire the lung images, the isotope and vector were admixed. Thus, the isotope marked the initial deposition location and the biodistribution for the rAAV2-GFP vector in the lungs of these monkeys. The vector particles were significantly smaller (20 nm diameter) [22Berns, K. I.1990. Parvoviridae and their replication. In Virology 2nd ed.B. N. Fieldset al., Eds. pp. 1743–1763. Raven Press, Ltd. New York.Google Scholar] than the saline-isotope droplets generated by the Microsprayer (∼ 22 μm diameter). Thus, the vector particles would have been in suspension in the saline-isotope solution and would have codistributed with the marker. In addition, uptake of the vector by the cells or absorption of the marker into the systemic circulation would have required more time than was needed to acquire the initial lung image. The dose administered per region was strikingly uniform (except for region 1) and reflected similar regional DNA transfer with some variability. The variation in gene transfer (Fig. 3B) could be explained by the time delay between dose delivery (t = 0) and DNA analysis (3 weeks later), mucociliary clearance, gravitational forces, or local mediators affecting viral uptake.rAAV2-mediated GFP transfer was detected minimally in some nontargeted tissues. Tissues in close proximity to the delivery site, such as the bronchial lymph nodes and oropharynx, tended to show gene transfer. Given the high initial doses of vector and our high transduction rates, this is not surprising. Transfer to gastrointestinal sites could be due to mucocilliary clearance of the vector followed by swallowing. The lack of transfer to the brain and reproductive organs is important in terms of the safety of pulmonary AAV gene therapy.Our data showed that rAAV2-mediated GFP expression increased when regional doses exceeded 3 × 109 i.u. Thus, maximizing the delivered dose is particularly important when using rAAV vectors because there appears to be a threshold deposition necessary for expression. These data suggest that future studies of rAAV pulmonary gene delivery and DNA transfer should use a starting dose that surpasses this threshold dose. Furthermore, direct administration of rAAV vector using a Microsprayer is a strategy to maximize delivery of high doses.Expression of rAAV2-GFP in the lungs of Rhesus macaques appears to be related to depositing a regional threshold dose exceeding 3 × 109 i.u. One way to achieve that dose is by bronchoscopic microspraying. Results from this study also suggest that the combination of optimizing lung delivery with bronchoscopic microspraying and high-titer production [23Monahan P. Samulski R. AAV vectors: Is clinical success on the horizon?.Gene Ther. 2000; 7: 24-30Crossref PubMed Scopus (262) Google Scholar] may advance the clinical applications of AAV pulmonary gene therapy for diseases such as cystic fibrosis.Materials and MethodsAnimalsJuvenile Rhesus macaques (three vector-treated, one control) were from the Johns Hopkins University breeding colony and housed according to Animal Care and Use Com