- Email: [email protected]
Immediate and Long-Term Safety of Recombinant Adeno-associated Virus Injection into the Nonhuman Primate Muscle
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
ARTICLE
Immediate and Long-Term Safety of Recombinant Adeno-associated Virus Injection into the Nonhuman Primate Muscle David Favre,1 Nathalie Provost,1 Véronique Blouin,1 Gilles Blancho,2 Yan Chérel,3 Anna Salvetti,1 and Philippe Moullier1,* 1
Laboratoire de Thérapie Génique, INSERM ERM-0105, CHU-Hotel DIEU, Bât. J. Monnet, 30 Avenue J. Monnet, 44035 Nantes Cedex 01, France 2 INSERM, U437, CHU-Hotel DIEU, Bât. J. Monnet, 30 Avenue J. Monnet, 44035 Nantes Cedex 01, France 3 Laboratoire d’Anatomie Pathologique, INRA UR 703, Ecole Nationale Vétérinaire, 44000 Nantes, France *To whom correspondence and reprint requests should be addressed. Fax: (33) 240087491. E-mail: [email protected].
Previous studies on distribution and toxicity of viral vectors administered in monkeys indicated that the nonhuman primate model has a reasonable predictive value for clinical applications. In this study, eight macaques were injected intramuscularly with recombinant adeno-associated virus (rAAV) at doses similar to those administered to hemophilia B patients, and followed to analyze the dissemination and shedding in biological samples and long-term persistence in distant organs. Following rAAV delivery, we found vector genome in various biological fluids for up to 6 days and infectious particles exclusively in the serum during the first 48–72 hours. rAAV sequences were detected in peripheral blood mononuclear cells (PBMC) for up to 10 months. At necropsy, 8 to 18 months after rAAV delivery, rAAV sequences were found in lymph nodes and livers but never in the gonads. Tissue examination, of liver in particular, showed no abnormalities. We concluded that during our experimental time frame, rAAV-mediated gene transfer into skeletal muscle of macaques seemed to be safe with respect to the recipient and the environment. However, it was associated with a transient viremia and the persistence of rAAV sequences in PBMC, lymph nodes, and liver, the long-term consequences of which remain unknown. Key words: gene therapy, adeno-associated virus, biodistribution, shedding, safety, skeletal muscle, nonhuman primate
INTRODUCTION Human adeno-associated virus type 2 (AAV-2) was engineered for use as a recombinant vector (rAAV) for in vivo gene transfer. AAV-2 is a helper-dependent human parvovirus that requires co-infection with adenovirus or herpesvirus for replication and virus assembly [1,2]. The virus has a stable capsid of approximately 25 nm in diameter harboring a 4.7-kb single-stranded DNA genome. The rAAV genome is composed of the two 145-base inverted terminal repeats (ITRs) flanking the transgene(s) and the transcriptional control elements. rAAVs are capable of efficient and prolonged transgene expression in several tissues including skeletal muscle, brain, liver, and retina [3]. On the basis of encouraging results obtained in mice and dogs, a clinical trial involving intramuscular injection of rAAV encoding human factor IX has been initiated [4–6]. Other potential clinical applications using rAAV are also being considered for the treatment of primary neuromuscular disorders, such as limb-girdle mus-
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00
cular dystrophy [7], and diseases in which muscle serves as an ectopic site for secretion of a therapeutic protein such as erythropoietin or ␣-1-antitrypsin [8–10]. The primary requirement for safe and successful gene therapy trials using rAAV vectors is the availability of comprehensive preclinical biodistribution data after vector administration. Although mice and rabbits were used to provide important safety studies for the ongoing hemophilia B clinical trial [6] (ORDA web site: http://www4.od.nih.gov/oba/rac/minutes/3-99RAC.htm), we believe that nonhuman primates represent a substantial improvement and a more relevant and informative preclinical model for rAAV safety assessment than rodents. More generally, previous studies showed that adenoviral vector distribution and toxicity in monkeys [11–13] closely correlated the clinical outcome [14,15]. As such, the nonhuman primate model has a reasonable predictive value for rAAV clinical developments. This study covered a period extending from 8 to 18 months following intramuscular (IM) injection of rAAV in
559
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
Although we have evidence for erythropoietin secretion upon doxycyclin administration to the animals, the transgene regulation aspects of this study was purposely excluded from this report for clarity. FIG. 1. Structure of rAAV vector plasmids. AAVcm-ET(LTR) and AAVcm-ET(CAG) vectors encode the cynomolgus erythropoietin cDNA (cmEpo) under the control of the doxycyclin-inducible tetO-CMV promoter and the rtTA chimeric protein under the control of the retroviral murine LTR or the CAG promoter, respectively. The rAAVcm-ET term alone designates both types of vectors. Thin arrows indicate the start transcription site; block arrows indicate the position of the primers used for the detection of rAAVcm-ET sequences. The size of the expected PCR product is indicated.
eight monkeys, providing an extensive database regarding the biodistribution and shedding of the viral particles in biological fluids and the long-term transduction of distant organs.
RESULTS Animals and rAAV Vector Doses Vectors encoding the cynomolgus erythropoietin cDNA (cmEpo) are represented in Fig. 1: the rAAVcm-ET vectors expressed the rtTA protein under the control of a constitutive promoter (LTR or CAG) and the cmEpo under the control of the doxycyclin-inducible tetO-CMV promoter [16]. We included eight animals in this study and divided them into three groups depending on the type of vector and on the dose and volume administered (Table 1). Following rAAV intramuscular administration, serum electrolytes, hematologic counts (before the doxycyclin inductions), liver function tests, serum creatinine, and blood urea nitrogen remained normal in all animals throughout the study (data not shown).
rAAV Dissemination and Shedding after Intramuscular rAAV Delivery The presence of the rAAV vector genome was assessed by PCR to detect the cmEpo DNA in several body fluids harvested from 30 minutes until 9 days post-injection. The sensitivity of the PCR assay was first evaluated by incubating a known number of viral particles (as measured by dot blot) with biological fluids before extracting the DNA. The result indicated that a threshold of 102 to 103 particles can be detected upon addition in culture medium, serum, and feces (Fig. 2A). PCR sensitivity was higher in urine, in which down to 10 rAAV particles could be consistently detected. PCR performed in the body fluids of all animals indicated that viral DNA could be detected as soon as 30 minutes in the serum after rAAV administration, and up to 6 days (Fig. 2B and Table 2). This finding was associated with shedding of the vector in the other body fluids (Table 2). The presence of infectious rAAV particles was simultaneously quantitated in the same samples by a modified replication center assay (mRCA). This test is based on the detection of replicating recombinant viral DNA in HeLaRC32 cells, which express the Rep proteins upon adenovirus infection [17]. The sensitivity of the mRCA was determined by adding the rAAV particles into either culture medium, which provided the standard value, or into different biological fluids. The results obtained indicated
TABLE 1: Characteristics of the animals and rAAV doses injected Group
Name
Sex/ speciesa
Age (years)
Weight (kg)
rAAV
Doseb (i.p./kg)
Volume (ml)
Current statusc
I
Mac 1
M/R
6
11
cmET (LTR)
5.0 ⫻ 108
5.0
sacrificed (8 months p.i.)
Mac 2
M/R
6
10
cmET (LTR)
5.0 ⫻ 108
5.0
sacrificed (8 months p.i.)
Mac 3
F/C
3.5
3
cmET (LTR)
2.5 ⫻ 109
25
sacrificed (18 months p.i.)
Mac 4
F/C
2.5
2.5
cmET (LTR)
8.0 ⫻ 109
26
sacrificed (18 months p.i.)
Mac 5
M/C
1.5
2
cmET (CAG)
3.0 ⫻ 109
2.4
alive
10
II
III
Mac 6
M/C
1.5
2
cmET (CAG)
1.0 ⫻ 10
2.4
alive
Mac 7
M/C
1.5
2
cmET (CAG)
4.0 ⫻ 109
2.4
alive
cmET (CAG)
4.0 ⫻ 10
2.4
alive
Mac 8
M/C
1.5
2
9
a
R, rhesus; C, cynomolgus. bi.p., Infectious particles. cp.i., Post rAAV injection.
560
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
A
ARTICLE
FIG. 2. Detection of AAV genome in body fluids. (A) Sensitivity of rAAVcm-ET vector sequence detection by PCR in biological fluids. Known amounts of rAAVcm-ET(LTR) genome particles, as determined by dot blot, were added either in medium or in biological samples as indicated, and viral DNA extracted. PCR was carried out using primers that amplified a 648-bp region between the tetO-CMV promoter and the cmEpo cDNA. After PCR amplification, reaction products were separated on a 2% agarose gel (top) and then transferred to a nylon membrane and hybridized to a cmEpo probe (bottom). The number of genome particles added is indicated at the top of each figure; (c+) refers to a positive control using 25 pg of the pAAVcmET vector plasmid. (B) Detection of rAAV genome particles in the serum. The serum of each animal was collected starting from 30 min (30’) until 9 days (9 d) post rAAVcmET(LTR) or rAAVcm-ET(CAG) administration (Table 1). Numbers on the top of each figure indicate at which time the sample was collected.
B
that the mRCA was 10- to 100-fold less sensitive when culture medium was replaced with biological fluids (Fig. 3A). After rAAV administration, rAAV infectious particles were unambiguously detected only in the serum of all the animals. A typical result is shown in Fig. 3B and the complete data are summarized in Fig. 4. Infectious particles were never detected in the serum of any animal past the first week (data not shown). Repeated PCR and mRCA analyses in all the biological fluids of group II and III animals were regularly performed past 1 month after vector administration. Both were negative. This finding indicated that although we had evidence that the injected muscles were still transduced, no mobilization of the vector was detected during the course of this study.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
rAAV Transduction of PBMC Because infectious rAAV were detected in serum, we investigated whether rAAV sequences could be found in PBMC at several time points. This was done by PCR analysis of rAAV sequence using genomic DNA from Ficoll-purified mononuclear cells. A positive signal was observed in animals from group I analyzed 8 months post-injection at the time of sacrifice (data not shown); from group II during the first 10 months (Fig. 5A); and from group III during the first 6 months after rAAV administration (data not shown), after which the rAAV signal was only occasionally detected. Next we sorted by FACS the mononuclear cells from two group III animals using either anti-CD2 (T cells) or anti-CD14 (monocytes, NK) antibody resulting in > 99% and approximately 90%
561
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
mus, thyroid, intestine, pancreas, and lymph nodes and used for PCR and Time post-injection histopathology. Hematoxylin and Fluid 30 min 6h 1 day 2 days 3–6 days >7 days eosin stained sections of each tissue Serum 3-4-6-7-8 5-6-7-8a all all 4-5-6-7-8 negative showed no abnormalities or inflamUrine n.d. 6-8a all except 7 3-4-6-8 4-8 negative mation (data not shown). Analysis of liver sections did not reveal evidence Feces n.d. n.d. 1-2-3-4 1-3-4 3-4 negative of cellular dysplasia. Sections from Saliva n.d. n.d. 3-4-8 3-4 negative negative transduced muscles were similar to Lacrymal n.d. 6a 3-4 2-4-8 4-8 negative untransduced muscles. Liver MRI were also performed on Mac 7 and Mac 8, Nasal n.d. 5-6a 3-4-6b 3-4-5-6-8b 3-4-5-8 negative 12 months post vector administration DNA was extracted from the different body fluids recovered at the indicated time point from each animal (indicated by and were normal. its number, see Table 1) and analyzed by PCR. The numbers indicate the animal (1 for Mac 1, 2 for Mac 2, and so on) found positive by PCR. n.d., Not done. rAAV sequences were searched by a Mac 1, 2, 3, and 4 not tested. PCR analysis using genomic DNA from b Mac 1 and 2 not tested. all of the tissues listed above. Figure 6 shows a typical result obtained with Mac 3 and Table 3 summarizes the purity, respectively. PCR analysis showed that both the complete results. As expected, rAAV sequences were found in all injected muscles. rAAV sequences were also CD2+ and CD14+ cell populations were positive for the detected in the liver (group II and Mac 5 and 6) and in presence of rAAV sequences (Fig. 5C). This indicated that the inguinal (group I and II animals), mesenteric, and trathe long-term positive PCR signal detected in PBMC after cheo-bronchial (group II animals) lymph nodes. The rAAV injection was derived at least from T cells. Finally, other organs and particularly the gonads were consisrAAV sequences were also detected by PCR using DNA tently negative. extracted from bone marrow cells of group III animals at 3 weeks and 4 months post rAAV administration (Fig. 5B), whereas control bone marrow obtained from Mac 8 DISCUSSION before rAAV injection was negative. We administered rAAV at a dose of from 5 ⫻ 108 to 1 ⫻ Analysis of rAAV Spread to Distant Organs 1010 infectious particles per kilogram of body weight (Table We analyzed the spread of rAAV to distant organs in 1). Considering that the genome particles to infectious group I and II animals that were sacrificed at 8 (Mac 1 particles ratio of our rAAV stocks was approximately 100, and 2) and 18 (Mac 3 and 4) months after rAAV admingroup II and III animals received doses equivalent to those istration. Group III animals were maintained alive and recently administered to patient in a clinical trial with laparotomy was performed 12 months post rAAV adminrAAV factor IX [6]. Also, our rAAV stocks were generated istration on Mac 5 and Mac 6 for liver biopsies with using the latest published procedure allowing greater simultaneous removal of the left testicle, in which vecpurity and lack of detectable wild-type AAV, as well as tor genome was searched. In all animals, no gross abnorinfectious adenovirus [18,19]. malities were noted in any internal organ or at the musThe first objective of our study was to analyze rAAV discle injection sites. Samples were taken from muscle, liver, semination and shedding, and to look for rAAV genome kidney, ovaries or testis, heart, lung, brain, spleen, thyparticles by PCR (Fig. 1) and infectious particles by mRCA. TABLE 2: Detection of rAAV vector sequences by PCR in body fluids
FIG. 3. Detection of infectious AAV by mRCA. (A) Detection of rAAVcm-ET infectious particles in biological fluids. The sensitivity of the mRCA in biological fluids (S, serum; F, feces; U, urine) was compared with the result obtained when rAAV was incubated in medium (M). The total number of infectious particles introduced in the well is indicated (top). (B) Detection of rAAVcm-ET(LTR) infectious particles in the serum of group II animals. The sera correspond to Mac 3 and Mac 4. mRCA was performed before rAAV delivery (0) and 30 minutes (30’) and 1, 2, and 5 days (1 d, 2 d, 5 d) after rAAV injection.
562
A
B
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
ARTICLE
Overall, our PCR data are in agreement with those reported from hemophilia B patients in whom positive PCR was found in serum, urine, and nasal fluid [6]. We extended this study by quantifying the infectious particles in body fluids and showed that infectious rAAV particles were only detected in serum for 48–72 hours after rAAV injection. It was recently shown in mice that rAAV can efficiently transduce slow myofibers and that it was at least partially related to the high expression of cytoplasmic heparan sulfate proteoglycan (HSP) [20]. Whether muscles that we injected are poorly expressing HSP and whether this issue has an impact on rAAV spread remain to be determined. In any case, the hematogenous spreadFIG. 4. Kinetics of rAAVcm-ET infectious particles in the serum of all eight animals. The num- ing of the vector following IM delivery had no ber of infectious particles/ml of serum is indicated for each animal at the different time points clinical adverse effects as judged by normal analyzed. All animals were tested at each time point (30’, 6 h, 1 d, 2 d), except where indiserum chemistries for liver and kidney funccated by an asterisk (*). tions. Also, because the injected volumes were different among the three groups, we cannot conclude on a possible vector shedding to vector dose correlation. The other biological fluids, such as The results of the PCR analysis indicated that following IM injection, rAAV particles can be detected in all body flu- urine and nasal samples, were consistently negative by ids for approximately 6 days (Fig. 2 and Table 2) suggest- mRCA. Because PCR and mRCA assays displayed similar ing the potential for spread into the environment. This sensitivity, these findings suggested that the rAAV partioutcome remained unchanged after pretreatment of the cles found in the various biological samples by PCR were samples with DNase I (data not shown), indicating that the not infectious. Previous reports in patients or in animal models sugPCR signal resulted from packaged rAAV sequences rather gested that wild-type AAV-2 sequences can be found by PCR than free rAAV DNA.
A
FIG. 5. Detection of rAAVcm-ET sequence in PBMC and bone marrow cells. Genomic DNA was extracted from group II animals PBMC (A) and bone marrow cells from group III animals (B) and analyzed by PCR (top) and Southern blot (lower) as indicated in the legend of Fig. 2. The number at the top of each lane indicates the time, in months, following rAAV injection at which the sample was harvested. (C) PCR results after fractionation of PBMC, from Mac 6 and Mac 8 at 5 and 2 months after rAAV injection, respectively. Each DNA sample was also positive for the amplification of cytochrome B sequence (data not shown). PCR performed using 25 pg of pAAVcm-ET vector plasmid or water served as positive (c+) and negative (c-) controls, respectively.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
563
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
FIG. 6. PCR analysis of rAAVcm-ET vector in distant organs. Genomic DNA was extracted from each tissue of Mac 3 at the time of sacrifice, 18 months after rAAV injection. As a control, each sample was also used for the amplification of a 350-bp cytochrome-B DNA fragment.
in blood cells, suggesting that they could constitute a “reservoir” for the virus [21,22]. We consistently detected rAAV sequences in PBMC of all the injected animals for at least 6 to 10 months. PBMC fractionation using the T cells’ CD2 or monocytes’ CD14 surface markers indicated the presence of the vector in T cells. Because CD14 cell sorting resulted in relative purity, we remain cautious of whether monocytes were actually transduced. Hernandez et al. [22] previously showed in a macaque model that IM injection of wildtype AAV-2 resulted in the detection of AAV sequences in PBMC 21 days post-injection. In our case, we detected vector DNA for a much longer period, although not by FISH analysis of interphase PBMC nuclei, probably due to the low number of transduced circulating cells. We also looked into whether the AAV vector in PBMC could be mobilized in vitro in the presence of wtAAV-2 and, as a helper, the adenovirus host range mutant AdHR405 [23] or the herpesvirus. In these conditions and despite numerous attempts, we
found no evidence for the production of infectious rAAV particles (data not shown). The second objective of our study was to document the rAAV spread to distant organs following IM injection. PCR analysis at necropsy (8 to 18 months post injection) showed that the transgene was easily detectable in the injected muscles, the liver, and lymph nodes (Fig. 6 and Table 3). No rAAV sequences were ever detected in the gonads. The spread of rAAV to distant organs was documented in nonhuman primates following instillation in the lung [24] and in rodents following intraportal, IM, or intracochlear injection [25–28]. Except for intracochlear injection, rAAV sequences were detected in several organs and particularly in the liver. This and our results uphold that the design of rAAV vectors expressing the transgene in a tissue-specific fashion is important from a safety point of view. Recently, M. Sands’s group reported the frequent onset of hepatocarcinomas, one year after intravenous
TABLE 3: Detection of rAAV vector sequences by PCR in different organs Tissues Animals
M
Li
LN
Lu
S
K
H
B
G
I
Mac 1
+
+
+
-
-
-
-
n.d.
-
-
Mac 2
+
+
+
-
-
-
-
n.d.
-
-
Mac 3
+
+
+
-
-
-
-
-
-
-
Mac 4
+
+
+
-
-
-
-
-
-
-
a
Mac 5
+
+
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
-
n.d.
Mac 6a
+
+
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
-
n.d.
M, rAAV injected muscle; Li, liver; LN, lymph nodes; Lu, lung; S, spleen; K, kidney; H, heart; B, brain; G, gonads; I, intestine; (+), positive; (-), negative; n.d., not done. a These results were obtained from liver and gonad biopsies carried out 12 months after rAAV injection.
564
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
injection of rAAV in murine mucopolysaccharidosis type VII neonates [29]. Although the maximum follow-up was limited to 18 months, our necropsy and MRI data support the safety of rAAV because livers were found to be macroscopically normal with no histologic evidence for dysplasia or tumor. Lastly, PCR signals found in lymph nodes from popliteal and inguinal areas homolateral to the injected muscle as well as mesenteric and tracheobronchial territories at necropsy in all (4/4) tested animals were unexpected. However, it is tempting to link these findings with those describing the long-term transduction of PBMC, resulting in a model in which the lymph node would be a preferential reservoir for rAAV-transduced resident mononuclear cells with occasional release into the circulation. The potential immunologic consequences of such a model remain to be appreciated, particularly with respect to transgene expression. Our study establishes that rAAV injection in the skeletal muscle at doses similar to that recently administered to hemophilia B patients is well tolerated with limited shedding of noninfectious rAAV. Nevertheless, the transient detection of infectious particles in the serum associated with the persistent transduction of PBMC, lymph nodes and the liver represents a safety concern for the recipient that has to be specifically addressed in the future. As clinical applications of rAAV are likely to expand rapidly, long-term follow-up studies on nonhuman primates should be pursued to fully appreciate the potential consequences of these observations.
MATERIALS
AND
METHODS
rAAV constructs and production. The rAAV constructs encoding the Macacca fascicularis (cynomolgus, cm) Epo cDNA were derived from plasmid pspER. This construct (provided by Delphine Bohl, Pasteur Institut, Paris) contained the cmEpo under the control of the tetO-CMV promoter [16] and, in the opposite orientation, an expression cassette encoding the reverse transactivator protein (rtTA) under the control of the retroviral MFG long-terminal repeat (LTR) [30]. A bidirectional SV40 polyadenylation signal was inserted between Epo and the rtTa transgenes. To construct the plasmid AAVcm-ET(LTR), the entire Op-CMV-Epo-rtTA-LTR DNA fragment (4349 bp) was excised using XhoI/ClaI, blunted, and inserted into psub201 between the SnaBI sites [31]. The plasmid AAVcm-ET(CAG) was derived from the previous construct by removing the 1.8-kb MFG LTR using EcoRI and PmlI and replacing it with the 1.1-kb CAG promoter [32]. rAAV was produced by cotransfecting 293 cells by the calcium phosphate method with the pDG [18] and the vector plasmids (25 mg and 12.5 g each per 15-cm plate, respectively). Six hours later, the cells were washed and incubated in DMEM with 5% FCS for 48 h. After harvesting, the cell pellet was resuspended in 50 mM Hepes (pH 7.6), 150 mM NaCl, and lysed by three cycles of freeze/thawing. rAAV particles were purified on a iodixanol gradient followed by a heparin column according to a described method [19]. The rAAV titer was determined by dot-blot analysis to provide the number of particles/ml based on the quantification of viral DNA and by mRCA to measure the number of infectious particles/ml. Both of these methods have been described [17]. Titers ranged from 1.109 i.p./ml (pAAVcm-ET(LTR)) to 2.1010 i.p./ml (pAAVcm-ET(CAG)) with a 10 to 100 physical to infectious particles ratio. Using the mRCA, neither infectious Ad5 nor Rep+ AAV particles could be detected in our vector stocks. AAV vector stocks produced in our vector core are routinely checked for Ad contamination by PCR and bacteriological testing for sterility.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
ARTICLE
Vector administration and specimen collection. Experiments were conducted on captive-bred rhesus and cynomolgus macaques purchased from the Centre de Primatologie, Rennemoulin, France, from the Centre de Primatologie, Strasbourg, France, and from Shamrock, Small Dole, England. Animals are described in Table 1. Neutralization assays carried out in the laboratory indicated that these animals did not have neutralizing antibodies against rAAV-2 particles. Serum, urine, feces, saliva, nasal, and lacrymal samples were regularly collected for 2 months before rAAV administration, and stored for the constitution of a baseline database. The protocol was approved by the Institutional Animal Care and Use Committee of the University. Anesthesia was performed with ketamine (10 mg/kg) before rAAV intramuscular administration, surgical biopsies, and collection of blood, nasal, saliva, and lacrymal samples. When biopsies were performed, buprenorphin (0.01 mg/kg) was administered twice a day for 2 days to avoid animal discomfort. The monkeys were divided into three groups based on the dose and volume administered (Table 1). Group I animals received five 1-ml injections of AAVcm-ET(LTR) into the left anterior tibialis muscle. Group II animals were given a total of 25 and 26 bilateral 1-ml injections of AAVcmET(LTR) in the tibialis anterior, gastrocnemius, quadriceps, and soleus muscles. Group III animals received six 400-ml injections of concentrated AAVcm-ET(CAG) into both anterior tibialis muscles. To enhance rAAV transduction, muscles were pretreated with clinical grade hyaluronidase (Hyaluronidase, Choay) IM, for a total dose of 1000 IU per muscle, 3 hours before vector delivery [33]. Analysis of rAAV DNA and infectious particles in body fluids. Serum, urine, feces, saliva, lacrymal, and nasal samples were repeatedly collected as soon as 30 min post injection and subsequently. Urine samples were filtered through a 0.22-m disposable filter. Feces and swabs were resuspended in DMEM medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma) and 1% penicillin/streptomycin (Gibco BRL, 5000 U/ml). Cleared supernatants were aliquoted and all samples frozen at –80⬚C before analysis. Vector genome in biological fluids was detected by PCR analysis, and the presence in the same samples of infectious rAAV particles was quantitated using the mRCA. For PCR analysis, rAAV DNA was extracted from 140 ml of samples using the Qiamp Viral RNA minikit (Qiagen, SA, France). Previous incubation of the sample with 10 U of DNase I (Boehringer) for 1 h at 37⬚C was performed only for urine. One tenth of the extraction (8 l) was then analyzed by PCR: the 5⬘ primer (5⬘-GAGTAGGCGTGTACGGTGGGAGG-3⬘) was located in the tetO-CMV promoter and the 3⬘ primer (5⬘GTGCAGCAGTGATGGTTCGGAG-3⬘) in the 3⬘ end of the cmEpo cDNA (Fig. 1). PCR-amplified vector sequence yielded a 648-bp fragment. For feces and urine samples, a control reaction containing the pre-injection sample spiked with vector plasmid (100 copies/g sample DNA) was also analyzed to confirm that neither urine nor feces inhibited the PCR reactions. After initial denaturation at 95⬚C for 2 min, 10 cycles were run at 95⬚C for 30 s, 70⬚C for 30 s, 72⬚C for 30 s, followed by 30 cycles at 95⬚C for 30 s, 60⬚C for 30 s, and 72⬚C for 30 s using Taq DNA polymerase (Promega) in a Perkin-Elmer thermocycler (PE, USA). Amplified products were analyzed by agarose gel electrophoresis followed by a transfer under alkaline conditions to a Hybond N+ membrane (Amersham). The membrane was hybridized to a 280-bp PvuII-AvaI fluorescein-labeled cmEpo probe (Amersham, Gene Images random prime labeling module) and incubated overnight at 65⬚C. The following day the membrane was processed according to the manufacturer’s protocol (Amersham, Gene Images CDPstar detection module) and exposed to autoradiography film. To detect rAAV infectious particles, minor modifications were introduced in the original mRCA protocol [17]. Briefly, 200 l of each biological fluid sample was added on 106 subconfluent HeLaRC32 cells for 2 hours. In some instances, samples (that is, feces) inhibited Ad5 infection of HeLaRC32 cells. For this reason, Ad5 was added at a MOI of 200 for 2 hours before the addition of each biological fluid sample. Two days later, cells were harvested and treated as described [17]. Replication centers were revealed using the 280-bp cmEpo probe described above. Extraction of genomic DNA from PBMC and other tissues. Mononuclear cells (2 ⫻ 107 cells) were isolated on Ficoll (Eurobio). Monocytes and NK
565
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
cells were sorted by FACS using an FITC-labeled anti-human CD14 antibody (clone MY4-FITC, Coulter Corporation, USA); T lymphocytes were sorted using phytoerythrin-labeled anti-human CD2 antibody (clone 39C1.5-PE, Immunotech, France). Total DNA was extracted from total PBMC and the sorted cells by lysis in a 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate (SDS) solution containing 500 g/ml proteinase K (Boehringer Mannheim). After overnight digest at 50⬚C, the DNA was extracted twice with phenol-chloroform, precipitated, and resuspended in 10 mM Tris, 1 mM EDTA (pH 8.0). In other tissue samples, genomic DNA was extracted using the same procedure as for PBMC, except the lysis step which was carried out in a 8 M urea, 10 mM EDTA, 300 mM NaCl, 10 mM Tris, 1% SDS buffer containing 500 mg proteinase K/ml overnight at 37⬚C. PCR analysis was performed using 750 ng DNA as described above. A control reaction was performed to detect cytochrome B sequence using two primers (5⬘-CCCCTCAGAATGATATTTGTCCTCA-3⬘/5⬘-CCATCCAACATCTCAGCATGATGAAA-3⬘) which amplified a 350-bp fragment.
ACKNOWLEDGMENTS We thank Jean-Michel Heard (Pasteur Institute, Paris) and Nick Muzyczka (University of Florida, Gainesville) for critically reading the manuscript and helpful discussions. This work was supported by the Association Française contre les Myopathies (AFM), Vaincre les Maladies Lysosomales (VML), the Association Nantaise de Thérapie Génique (ANTG), and the Fondation pour la Thérapie Génique en Pays de la Loire. D.F. was supported by a fellowship from the Ministère de la Recherche. RECEIVED FOR PUBLICATION MAY 31; ACCEPTED SEPTEMBER 28, 2001.
REFERENCES 1. Muzyczka, N. (1992). Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr. Top. Microbiol. Immunol. 158: 97–129. 2. Berns, K. I., and Giraud, C. (1996). Biology of adeno-associated virus. Curr. Top. Microbiol. Immunol. 218: 1–23. 3. Snyder, R. O. (1999). Adeno-associated virus-mediated gene delivery. J. Gene Med. 1: 166–175. 4. Herzog, R. W., et al. (1997). Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc. Natl. Acad. Sci. USA 94: 5804–5809. 5. Herzog, R. W., et al. (1999). Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat. Med. 5: 56–63. 6. Kay, M. A., et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24: 257–261. 7. Xiao, X., et al. (2000). Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy. J. Virol. 74: 1436–1442. 8. Bohl, D., Salvetti, A., Moullier, P., and Heard, J. M. (1998). Control of erythropoietin delivery by doxycyclin in mice after intramuscular injection of adeno-associated vector. Blood 92: 1512–1517. 9. Bohl, D., Bosch, A., Cardona, A., Salvetti, A., and Heard, J. M. (2000). Improvement of erythropoiesis in -thalassemic mice by continuous erythropoietin delivery from muscle. Blood 95: 2793–2798.
566
10. Song, S., et al. (1998). Sustained secretion of human ␣-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc. Natl. Acad. Sci. USA 95: 14384–14388. 11. Simon, R. H., et al. (1993). Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. Hum. Gene Ther. 4: 771–780. 12. Sene, C., et al. (1995). Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates. Hum. Gene Ther. 6: 1587–1593. 13. Nunes, F. A., Furth, E. E., Wilson, J. M., and Raper, S. E. (1999). Gene transfer into the liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration. Hum. Gene Ther. 10: 2515–2526. 14. Crystal, R. G., et al. (1994). Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat. Genet. 8: 42–51. 15. Bellon, G., et al. (1997). Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial. Hum. Gene Ther. 8: 15–25. 16. Gossen, M., and Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracyclin-responsive promoters. Proc. Natl. Acad. Sci. USA 89: 5547–5551. 17. Salvetti, A., et al. (1998). Factors influencing recombinant adeno-associated virus production. Hum. Gene Ther. 9: 695–706. 18. Grimm, D., Kern, A., Rittner, K., and Kleinschmidt, J. (1998). Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum. Gene Ther. 9: 2745–2760. 19. Zolotukhin, S., et al. (1999). Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6: 973–985. 20. Pruchnic, R., et al. (2000). The use of adeno-associated virus to circumvent the maturation-dependent viral transduction of muscle fibers. Hum. Gene Ther. 11: 521–536. 21. Grossman, Z., et al. (1992). Detection of adeno-associated virus type 2 in human peripheral blood cells. J. Gen. Virol. 73: 961–966. 22. Hernandez, Y. J., et al. (1999). Latent adeno-associated virus infection elicits humoral but not cell-mediated immune responses in a nonhuman primate model. J. Virol. 73: 8549–8558. 23. Afione, S. A., et al. (1996). In vivo model of adeno-associated virus vector persistence and rescue. J. Virol. 70: 3235–3241. 24. Conrad, C. K., et al. (1996). Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther. 3: 658–668. 25. Monahan, P. E., et al. (1998). Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia. Gene Ther. 5: 40–49. 26. Snyder, R. O., et al. (1999). Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat. Med. 5: 64–70. 27. Soochuen, T. K., Pettis, R. M., Mhatre, A. N., and Lalwani, A. K. (2000). Safety of adenoassociated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae. Mol. Ther. 2: 368–373, doi:10.1006/mthe.2000.0129 28. Nathwani, A. C., et al. (2001). Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA. Blood 97: 1258–1265. 29. Donsante, A., et al. (2001). Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors. Gene Ther. 8: 1343–1346. 30. Dranoff, G., et al. (1993). Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long lasting anti-tumor immunity. Proc. Natl. Acad. Sci. USA 90: 3539–3543. 31. Samulski, R. J., Chang, L. S., and Shenk, T. (1989). Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol. 63: 3822–3828. 32. Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193–199. 33. Favre, D., et al. (2000). Hyaluronidase enhances recombinant adeno-associated virus (rAAV)-mediated gene transfer in the rat skeletal muscle. Gene Ther. 7: 1417–1420.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
ARTICLE
Immediate and Long-Term Safety of Recombinant Adeno-associated Virus Injection into the Nonhuman Primate Muscle David Favre,1 Nathalie Provost,1 Véronique Blouin,1 Gilles Blancho,2 Yan Chérel,3 Anna Salvetti,1 and Philippe Moullier1,* 1
Laboratoire de Thérapie Génique, INSERM ERM-0105, CHU-Hotel DIEU, Bât. J. Monnet, 30 Avenue J. Monnet, 44035 Nantes Cedex 01, France 2 INSERM, U437, CHU-Hotel DIEU, Bât. J. Monnet, 30 Avenue J. Monnet, 44035 Nantes Cedex 01, France 3 Laboratoire d’Anatomie Pathologique, INRA UR 703, Ecole Nationale Vétérinaire, 44000 Nantes, France *To whom correspondence and reprint requests should be addressed. Fax: (33) 240087491. E-mail: [email protected].
Previous studies on distribution and toxicity of viral vectors administered in monkeys indicated that the nonhuman primate model has a reasonable predictive value for clinical applications. In this study, eight macaques were injected intramuscularly with recombinant adeno-associated virus (rAAV) at doses similar to those administered to hemophilia B patients, and followed to analyze the dissemination and shedding in biological samples and long-term persistence in distant organs. Following rAAV delivery, we found vector genome in various biological fluids for up to 6 days and infectious particles exclusively in the serum during the first 48–72 hours. rAAV sequences were detected in peripheral blood mononuclear cells (PBMC) for up to 10 months. At necropsy, 8 to 18 months after rAAV delivery, rAAV sequences were found in lymph nodes and livers but never in the gonads. Tissue examination, of liver in particular, showed no abnormalities. We concluded that during our experimental time frame, rAAV-mediated gene transfer into skeletal muscle of macaques seemed to be safe with respect to the recipient and the environment. However, it was associated with a transient viremia and the persistence of rAAV sequences in PBMC, lymph nodes, and liver, the long-term consequences of which remain unknown. Key words: gene therapy, adeno-associated virus, biodistribution, shedding, safety, skeletal muscle, nonhuman primate
INTRODUCTION Human adeno-associated virus type 2 (AAV-2) was engineered for use as a recombinant vector (rAAV) for in vivo gene transfer. AAV-2 is a helper-dependent human parvovirus that requires co-infection with adenovirus or herpesvirus for replication and virus assembly [1,2]. The virus has a stable capsid of approximately 25 nm in diameter harboring a 4.7-kb single-stranded DNA genome. The rAAV genome is composed of the two 145-base inverted terminal repeats (ITRs) flanking the transgene(s) and the transcriptional control elements. rAAVs are capable of efficient and prolonged transgene expression in several tissues including skeletal muscle, brain, liver, and retina [3]. On the basis of encouraging results obtained in mice and dogs, a clinical trial involving intramuscular injection of rAAV encoding human factor IX has been initiated [4–6]. Other potential clinical applications using rAAV are also being considered for the treatment of primary neuromuscular disorders, such as limb-girdle mus-
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00
cular dystrophy [7], and diseases in which muscle serves as an ectopic site for secretion of a therapeutic protein such as erythropoietin or ␣-1-antitrypsin [8–10]. The primary requirement for safe and successful gene therapy trials using rAAV vectors is the availability of comprehensive preclinical biodistribution data after vector administration. Although mice and rabbits were used to provide important safety studies for the ongoing hemophilia B clinical trial [6] (ORDA web site: http://www4.od.nih.gov/oba/rac/minutes/3-99RAC.htm), we believe that nonhuman primates represent a substantial improvement and a more relevant and informative preclinical model for rAAV safety assessment than rodents. More generally, previous studies showed that adenoviral vector distribution and toxicity in monkeys [11–13] closely correlated the clinical outcome [14,15]. As such, the nonhuman primate model has a reasonable predictive value for rAAV clinical developments. This study covered a period extending from 8 to 18 months following intramuscular (IM) injection of rAAV in
559
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
Although we have evidence for erythropoietin secretion upon doxycyclin administration to the animals, the transgene regulation aspects of this study was purposely excluded from this report for clarity. FIG. 1. Structure of rAAV vector plasmids. AAVcm-ET(LTR) and AAVcm-ET(CAG) vectors encode the cynomolgus erythropoietin cDNA (cmEpo) under the control of the doxycyclin-inducible tetO-CMV promoter and the rtTA chimeric protein under the control of the retroviral murine LTR or the CAG promoter, respectively. The rAAVcm-ET term alone designates both types of vectors. Thin arrows indicate the start transcription site; block arrows indicate the position of the primers used for the detection of rAAVcm-ET sequences. The size of the expected PCR product is indicated.
eight monkeys, providing an extensive database regarding the biodistribution and shedding of the viral particles in biological fluids and the long-term transduction of distant organs.
RESULTS Animals and rAAV Vector Doses Vectors encoding the cynomolgus erythropoietin cDNA (cmEpo) are represented in Fig. 1: the rAAVcm-ET vectors expressed the rtTA protein under the control of a constitutive promoter (LTR or CAG) and the cmEpo under the control of the doxycyclin-inducible tetO-CMV promoter [16]. We included eight animals in this study and divided them into three groups depending on the type of vector and on the dose and volume administered (Table 1). Following rAAV intramuscular administration, serum electrolytes, hematologic counts (before the doxycyclin inductions), liver function tests, serum creatinine, and blood urea nitrogen remained normal in all animals throughout the study (data not shown).
rAAV Dissemination and Shedding after Intramuscular rAAV Delivery The presence of the rAAV vector genome was assessed by PCR to detect the cmEpo DNA in several body fluids harvested from 30 minutes until 9 days post-injection. The sensitivity of the PCR assay was first evaluated by incubating a known number of viral particles (as measured by dot blot) with biological fluids before extracting the DNA. The result indicated that a threshold of 102 to 103 particles can be detected upon addition in culture medium, serum, and feces (Fig. 2A). PCR sensitivity was higher in urine, in which down to 10 rAAV particles could be consistently detected. PCR performed in the body fluids of all animals indicated that viral DNA could be detected as soon as 30 minutes in the serum after rAAV administration, and up to 6 days (Fig. 2B and Table 2). This finding was associated with shedding of the vector in the other body fluids (Table 2). The presence of infectious rAAV particles was simultaneously quantitated in the same samples by a modified replication center assay (mRCA). This test is based on the detection of replicating recombinant viral DNA in HeLaRC32 cells, which express the Rep proteins upon adenovirus infection [17]. The sensitivity of the mRCA was determined by adding the rAAV particles into either culture medium, which provided the standard value, or into different biological fluids. The results obtained indicated
TABLE 1: Characteristics of the animals and rAAV doses injected Group
Name
Sex/ speciesa
Age (years)
Weight (kg)
rAAV
Doseb (i.p./kg)
Volume (ml)
Current statusc
I
Mac 1
M/R
6
11
cmET (LTR)
5.0 ⫻ 108
5.0
sacrificed (8 months p.i.)
Mac 2
M/R
6
10
cmET (LTR)
5.0 ⫻ 108
5.0
sacrificed (8 months p.i.)
Mac 3
F/C
3.5
3
cmET (LTR)
2.5 ⫻ 109
25
sacrificed (18 months p.i.)
Mac 4
F/C
2.5
2.5
cmET (LTR)
8.0 ⫻ 109
26
sacrificed (18 months p.i.)
Mac 5
M/C
1.5
2
cmET (CAG)
3.0 ⫻ 109
2.4
alive
10
II
III
Mac 6
M/C
1.5
2
cmET (CAG)
1.0 ⫻ 10
2.4
alive
Mac 7
M/C
1.5
2
cmET (CAG)
4.0 ⫻ 109
2.4
alive
cmET (CAG)
4.0 ⫻ 10
2.4
alive
Mac 8
M/C
1.5
2
9
a
R, rhesus; C, cynomolgus. bi.p., Infectious particles. cp.i., Post rAAV injection.
560
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
A
ARTICLE
FIG. 2. Detection of AAV genome in body fluids. (A) Sensitivity of rAAVcm-ET vector sequence detection by PCR in biological fluids. Known amounts of rAAVcm-ET(LTR) genome particles, as determined by dot blot, were added either in medium or in biological samples as indicated, and viral DNA extracted. PCR was carried out using primers that amplified a 648-bp region between the tetO-CMV promoter and the cmEpo cDNA. After PCR amplification, reaction products were separated on a 2% agarose gel (top) and then transferred to a nylon membrane and hybridized to a cmEpo probe (bottom). The number of genome particles added is indicated at the top of each figure; (c+) refers to a positive control using 25 pg of the pAAVcmET vector plasmid. (B) Detection of rAAV genome particles in the serum. The serum of each animal was collected starting from 30 min (30’) until 9 days (9 d) post rAAVcmET(LTR) or rAAVcm-ET(CAG) administration (Table 1). Numbers on the top of each figure indicate at which time the sample was collected.
B
that the mRCA was 10- to 100-fold less sensitive when culture medium was replaced with biological fluids (Fig. 3A). After rAAV administration, rAAV infectious particles were unambiguously detected only in the serum of all the animals. A typical result is shown in Fig. 3B and the complete data are summarized in Fig. 4. Infectious particles were never detected in the serum of any animal past the first week (data not shown). Repeated PCR and mRCA analyses in all the biological fluids of group II and III animals were regularly performed past 1 month after vector administration. Both were negative. This finding indicated that although we had evidence that the injected muscles were still transduced, no mobilization of the vector was detected during the course of this study.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
rAAV Transduction of PBMC Because infectious rAAV were detected in serum, we investigated whether rAAV sequences could be found in PBMC at several time points. This was done by PCR analysis of rAAV sequence using genomic DNA from Ficoll-purified mononuclear cells. A positive signal was observed in animals from group I analyzed 8 months post-injection at the time of sacrifice (data not shown); from group II during the first 10 months (Fig. 5A); and from group III during the first 6 months after rAAV administration (data not shown), after which the rAAV signal was only occasionally detected. Next we sorted by FACS the mononuclear cells from two group III animals using either anti-CD2 (T cells) or anti-CD14 (monocytes, NK) antibody resulting in > 99% and approximately 90%
561
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
mus, thyroid, intestine, pancreas, and lymph nodes and used for PCR and Time post-injection histopathology. Hematoxylin and Fluid 30 min 6h 1 day 2 days 3–6 days >7 days eosin stained sections of each tissue Serum 3-4-6-7-8 5-6-7-8a all all 4-5-6-7-8 negative showed no abnormalities or inflamUrine n.d. 6-8a all except 7 3-4-6-8 4-8 negative mation (data not shown). Analysis of liver sections did not reveal evidence Feces n.d. n.d. 1-2-3-4 1-3-4 3-4 negative of cellular dysplasia. Sections from Saliva n.d. n.d. 3-4-8 3-4 negative negative transduced muscles were similar to Lacrymal n.d. 6a 3-4 2-4-8 4-8 negative untransduced muscles. Liver MRI were also performed on Mac 7 and Mac 8, Nasal n.d. 5-6a 3-4-6b 3-4-5-6-8b 3-4-5-8 negative 12 months post vector administration DNA was extracted from the different body fluids recovered at the indicated time point from each animal (indicated by and were normal. its number, see Table 1) and analyzed by PCR. The numbers indicate the animal (1 for Mac 1, 2 for Mac 2, and so on) found positive by PCR. n.d., Not done. rAAV sequences were searched by a Mac 1, 2, 3, and 4 not tested. PCR analysis using genomic DNA from b Mac 1 and 2 not tested. all of the tissues listed above. Figure 6 shows a typical result obtained with Mac 3 and Table 3 summarizes the purity, respectively. PCR analysis showed that both the complete results. As expected, rAAV sequences were found in all injected muscles. rAAV sequences were also CD2+ and CD14+ cell populations were positive for the detected in the liver (group II and Mac 5 and 6) and in presence of rAAV sequences (Fig. 5C). This indicated that the inguinal (group I and II animals), mesenteric, and trathe long-term positive PCR signal detected in PBMC after cheo-bronchial (group II animals) lymph nodes. The rAAV injection was derived at least from T cells. Finally, other organs and particularly the gonads were consisrAAV sequences were also detected by PCR using DNA tently negative. extracted from bone marrow cells of group III animals at 3 weeks and 4 months post rAAV administration (Fig. 5B), whereas control bone marrow obtained from Mac 8 DISCUSSION before rAAV injection was negative. We administered rAAV at a dose of from 5 ⫻ 108 to 1 ⫻ Analysis of rAAV Spread to Distant Organs 1010 infectious particles per kilogram of body weight (Table We analyzed the spread of rAAV to distant organs in 1). Considering that the genome particles to infectious group I and II animals that were sacrificed at 8 (Mac 1 particles ratio of our rAAV stocks was approximately 100, and 2) and 18 (Mac 3 and 4) months after rAAV admingroup II and III animals received doses equivalent to those istration. Group III animals were maintained alive and recently administered to patient in a clinical trial with laparotomy was performed 12 months post rAAV adminrAAV factor IX [6]. Also, our rAAV stocks were generated istration on Mac 5 and Mac 6 for liver biopsies with using the latest published procedure allowing greater simultaneous removal of the left testicle, in which vecpurity and lack of detectable wild-type AAV, as well as tor genome was searched. In all animals, no gross abnorinfectious adenovirus [18,19]. malities were noted in any internal organ or at the musThe first objective of our study was to analyze rAAV discle injection sites. Samples were taken from muscle, liver, semination and shedding, and to look for rAAV genome kidney, ovaries or testis, heart, lung, brain, spleen, thyparticles by PCR (Fig. 1) and infectious particles by mRCA. TABLE 2: Detection of rAAV vector sequences by PCR in body fluids
FIG. 3. Detection of infectious AAV by mRCA. (A) Detection of rAAVcm-ET infectious particles in biological fluids. The sensitivity of the mRCA in biological fluids (S, serum; F, feces; U, urine) was compared with the result obtained when rAAV was incubated in medium (M). The total number of infectious particles introduced in the well is indicated (top). (B) Detection of rAAVcm-ET(LTR) infectious particles in the serum of group II animals. The sera correspond to Mac 3 and Mac 4. mRCA was performed before rAAV delivery (0) and 30 minutes (30’) and 1, 2, and 5 days (1 d, 2 d, 5 d) after rAAV injection.
562
A
B
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
ARTICLE
Overall, our PCR data are in agreement with those reported from hemophilia B patients in whom positive PCR was found in serum, urine, and nasal fluid [6]. We extended this study by quantifying the infectious particles in body fluids and showed that infectious rAAV particles were only detected in serum for 48–72 hours after rAAV injection. It was recently shown in mice that rAAV can efficiently transduce slow myofibers and that it was at least partially related to the high expression of cytoplasmic heparan sulfate proteoglycan (HSP) [20]. Whether muscles that we injected are poorly expressing HSP and whether this issue has an impact on rAAV spread remain to be determined. In any case, the hematogenous spreadFIG. 4. Kinetics of rAAVcm-ET infectious particles in the serum of all eight animals. The num- ing of the vector following IM delivery had no ber of infectious particles/ml of serum is indicated for each animal at the different time points clinical adverse effects as judged by normal analyzed. All animals were tested at each time point (30’, 6 h, 1 d, 2 d), except where indiserum chemistries for liver and kidney funccated by an asterisk (*). tions. Also, because the injected volumes were different among the three groups, we cannot conclude on a possible vector shedding to vector dose correlation. The other biological fluids, such as The results of the PCR analysis indicated that following IM injection, rAAV particles can be detected in all body flu- urine and nasal samples, were consistently negative by ids for approximately 6 days (Fig. 2 and Table 2) suggest- mRCA. Because PCR and mRCA assays displayed similar ing the potential for spread into the environment. This sensitivity, these findings suggested that the rAAV partioutcome remained unchanged after pretreatment of the cles found in the various biological samples by PCR were samples with DNase I (data not shown), indicating that the not infectious. Previous reports in patients or in animal models sugPCR signal resulted from packaged rAAV sequences rather gested that wild-type AAV-2 sequences can be found by PCR than free rAAV DNA.
A
FIG. 5. Detection of rAAVcm-ET sequence in PBMC and bone marrow cells. Genomic DNA was extracted from group II animals PBMC (A) and bone marrow cells from group III animals (B) and analyzed by PCR (top) and Southern blot (lower) as indicated in the legend of Fig. 2. The number at the top of each lane indicates the time, in months, following rAAV injection at which the sample was harvested. (C) PCR results after fractionation of PBMC, from Mac 6 and Mac 8 at 5 and 2 months after rAAV injection, respectively. Each DNA sample was also positive for the amplification of cytochrome B sequence (data not shown). PCR performed using 25 pg of pAAVcm-ET vector plasmid or water served as positive (c+) and negative (c-) controls, respectively.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
563
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
FIG. 6. PCR analysis of rAAVcm-ET vector in distant organs. Genomic DNA was extracted from each tissue of Mac 3 at the time of sacrifice, 18 months after rAAV injection. As a control, each sample was also used for the amplification of a 350-bp cytochrome-B DNA fragment.
in blood cells, suggesting that they could constitute a “reservoir” for the virus [21,22]. We consistently detected rAAV sequences in PBMC of all the injected animals for at least 6 to 10 months. PBMC fractionation using the T cells’ CD2 or monocytes’ CD14 surface markers indicated the presence of the vector in T cells. Because CD14 cell sorting resulted in relative purity, we remain cautious of whether monocytes were actually transduced. Hernandez et al. [22] previously showed in a macaque model that IM injection of wildtype AAV-2 resulted in the detection of AAV sequences in PBMC 21 days post-injection. In our case, we detected vector DNA for a much longer period, although not by FISH analysis of interphase PBMC nuclei, probably due to the low number of transduced circulating cells. We also looked into whether the AAV vector in PBMC could be mobilized in vitro in the presence of wtAAV-2 and, as a helper, the adenovirus host range mutant AdHR405 [23] or the herpesvirus. In these conditions and despite numerous attempts, we
found no evidence for the production of infectious rAAV particles (data not shown). The second objective of our study was to document the rAAV spread to distant organs following IM injection. PCR analysis at necropsy (8 to 18 months post injection) showed that the transgene was easily detectable in the injected muscles, the liver, and lymph nodes (Fig. 6 and Table 3). No rAAV sequences were ever detected in the gonads. The spread of rAAV to distant organs was documented in nonhuman primates following instillation in the lung [24] and in rodents following intraportal, IM, or intracochlear injection [25–28]. Except for intracochlear injection, rAAV sequences were detected in several organs and particularly in the liver. This and our results uphold that the design of rAAV vectors expressing the transgene in a tissue-specific fashion is important from a safety point of view. Recently, M. Sands’s group reported the frequent onset of hepatocarcinomas, one year after intravenous
TABLE 3: Detection of rAAV vector sequences by PCR in different organs Tissues Animals
M
Li
LN
Lu
S
K
H
B
G
I
Mac 1
+
+
+
-
-
-
-
n.d.
-
-
Mac 2
+
+
+
-
-
-
-
n.d.
-
-
Mac 3
+
+
+
-
-
-
-
-
-
-
Mac 4
+
+
+
-
-
-
-
-
-
-
a
Mac 5
+
+
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
-
n.d.
Mac 6a
+
+
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
-
n.d.
M, rAAV injected muscle; Li, liver; LN, lymph nodes; Lu, lung; S, spleen; K, kidney; H, heart; B, brain; G, gonads; I, intestine; (+), positive; (-), negative; n.d., not done. a These results were obtained from liver and gonad biopsies carried out 12 months after rAAV injection.
564
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
injection of rAAV in murine mucopolysaccharidosis type VII neonates [29]. Although the maximum follow-up was limited to 18 months, our necropsy and MRI data support the safety of rAAV because livers were found to be macroscopically normal with no histologic evidence for dysplasia or tumor. Lastly, PCR signals found in lymph nodes from popliteal and inguinal areas homolateral to the injected muscle as well as mesenteric and tracheobronchial territories at necropsy in all (4/4) tested animals were unexpected. However, it is tempting to link these findings with those describing the long-term transduction of PBMC, resulting in a model in which the lymph node would be a preferential reservoir for rAAV-transduced resident mononuclear cells with occasional release into the circulation. The potential immunologic consequences of such a model remain to be appreciated, particularly with respect to transgene expression. Our study establishes that rAAV injection in the skeletal muscle at doses similar to that recently administered to hemophilia B patients is well tolerated with limited shedding of noninfectious rAAV. Nevertheless, the transient detection of infectious particles in the serum associated with the persistent transduction of PBMC, lymph nodes and the liver represents a safety concern for the recipient that has to be specifically addressed in the future. As clinical applications of rAAV are likely to expand rapidly, long-term follow-up studies on nonhuman primates should be pursued to fully appreciate the potential consequences of these observations.
MATERIALS
AND
METHODS
rAAV constructs and production. The rAAV constructs encoding the Macacca fascicularis (cynomolgus, cm) Epo cDNA were derived from plasmid pspER. This construct (provided by Delphine Bohl, Pasteur Institut, Paris) contained the cmEpo under the control of the tetO-CMV promoter [16] and, in the opposite orientation, an expression cassette encoding the reverse transactivator protein (rtTA) under the control of the retroviral MFG long-terminal repeat (LTR) [30]. A bidirectional SV40 polyadenylation signal was inserted between Epo and the rtTa transgenes. To construct the plasmid AAVcm-ET(LTR), the entire Op-CMV-Epo-rtTA-LTR DNA fragment (4349 bp) was excised using XhoI/ClaI, blunted, and inserted into psub201 between the SnaBI sites [31]. The plasmid AAVcm-ET(CAG) was derived from the previous construct by removing the 1.8-kb MFG LTR using EcoRI and PmlI and replacing it with the 1.1-kb CAG promoter [32]. rAAV was produced by cotransfecting 293 cells by the calcium phosphate method with the pDG [18] and the vector plasmids (25 mg and 12.5 g each per 15-cm plate, respectively). Six hours later, the cells were washed and incubated in DMEM with 5% FCS for 48 h. After harvesting, the cell pellet was resuspended in 50 mM Hepes (pH 7.6), 150 mM NaCl, and lysed by three cycles of freeze/thawing. rAAV particles were purified on a iodixanol gradient followed by a heparin column according to a described method [19]. The rAAV titer was determined by dot-blot analysis to provide the number of particles/ml based on the quantification of viral DNA and by mRCA to measure the number of infectious particles/ml. Both of these methods have been described [17]. Titers ranged from 1.109 i.p./ml (pAAVcm-ET(LTR)) to 2.1010 i.p./ml (pAAVcm-ET(CAG)) with a 10 to 100 physical to infectious particles ratio. Using the mRCA, neither infectious Ad5 nor Rep+ AAV particles could be detected in our vector stocks. AAV vector stocks produced in our vector core are routinely checked for Ad contamination by PCR and bacteriological testing for sterility.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy
ARTICLE
Vector administration and specimen collection. Experiments were conducted on captive-bred rhesus and cynomolgus macaques purchased from the Centre de Primatologie, Rennemoulin, France, from the Centre de Primatologie, Strasbourg, France, and from Shamrock, Small Dole, England. Animals are described in Table 1. Neutralization assays carried out in the laboratory indicated that these animals did not have neutralizing antibodies against rAAV-2 particles. Serum, urine, feces, saliva, nasal, and lacrymal samples were regularly collected for 2 months before rAAV administration, and stored for the constitution of a baseline database. The protocol was approved by the Institutional Animal Care and Use Committee of the University. Anesthesia was performed with ketamine (10 mg/kg) before rAAV intramuscular administration, surgical biopsies, and collection of blood, nasal, saliva, and lacrymal samples. When biopsies were performed, buprenorphin (0.01 mg/kg) was administered twice a day for 2 days to avoid animal discomfort. The monkeys were divided into three groups based on the dose and volume administered (Table 1). Group I animals received five 1-ml injections of AAVcm-ET(LTR) into the left anterior tibialis muscle. Group II animals were given a total of 25 and 26 bilateral 1-ml injections of AAVcmET(LTR) in the tibialis anterior, gastrocnemius, quadriceps, and soleus muscles. Group III animals received six 400-ml injections of concentrated AAVcm-ET(CAG) into both anterior tibialis muscles. To enhance rAAV transduction, muscles were pretreated with clinical grade hyaluronidase (Hyaluronidase, Choay) IM, for a total dose of 1000 IU per muscle, 3 hours before vector delivery [33]. Analysis of rAAV DNA and infectious particles in body fluids. Serum, urine, feces, saliva, lacrymal, and nasal samples were repeatedly collected as soon as 30 min post injection and subsequently. Urine samples were filtered through a 0.22-m disposable filter. Feces and swabs were resuspended in DMEM medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sigma) and 1% penicillin/streptomycin (Gibco BRL, 5000 U/ml). Cleared supernatants were aliquoted and all samples frozen at –80⬚C before analysis. Vector genome in biological fluids was detected by PCR analysis, and the presence in the same samples of infectious rAAV particles was quantitated using the mRCA. For PCR analysis, rAAV DNA was extracted from 140 ml of samples using the Qiamp Viral RNA minikit (Qiagen, SA, France). Previous incubation of the sample with 10 U of DNase I (Boehringer) for 1 h at 37⬚C was performed only for urine. One tenth of the extraction (8 l) was then analyzed by PCR: the 5⬘ primer (5⬘-GAGTAGGCGTGTACGGTGGGAGG-3⬘) was located in the tetO-CMV promoter and the 3⬘ primer (5⬘GTGCAGCAGTGATGGTTCGGAG-3⬘) in the 3⬘ end of the cmEpo cDNA (Fig. 1). PCR-amplified vector sequence yielded a 648-bp fragment. For feces and urine samples, a control reaction containing the pre-injection sample spiked with vector plasmid (100 copies/g sample DNA) was also analyzed to confirm that neither urine nor feces inhibited the PCR reactions. After initial denaturation at 95⬚C for 2 min, 10 cycles were run at 95⬚C for 30 s, 70⬚C for 30 s, 72⬚C for 30 s, followed by 30 cycles at 95⬚C for 30 s, 60⬚C for 30 s, and 72⬚C for 30 s using Taq DNA polymerase (Promega) in a Perkin-Elmer thermocycler (PE, USA). Amplified products were analyzed by agarose gel electrophoresis followed by a transfer under alkaline conditions to a Hybond N+ membrane (Amersham). The membrane was hybridized to a 280-bp PvuII-AvaI fluorescein-labeled cmEpo probe (Amersham, Gene Images random prime labeling module) and incubated overnight at 65⬚C. The following day the membrane was processed according to the manufacturer’s protocol (Amersham, Gene Images CDPstar detection module) and exposed to autoradiography film. To detect rAAV infectious particles, minor modifications were introduced in the original mRCA protocol [17]. Briefly, 200 l of each biological fluid sample was added on 106 subconfluent HeLaRC32 cells for 2 hours. In some instances, samples (that is, feces) inhibited Ad5 infection of HeLaRC32 cells. For this reason, Ad5 was added at a MOI of 200 for 2 hours before the addition of each biological fluid sample. Two days later, cells were harvested and treated as described [17]. Replication centers were revealed using the 280-bp cmEpo probe described above. Extraction of genomic DNA from PBMC and other tissues. Mononuclear cells (2 ⫻ 107 cells) were isolated on Ficoll (Eurobio). Monocytes and NK
565
ARTICLE
doi:10.1006/mthe.2001.0494, available online at http://www.idealibrary.com on IDEAL
cells were sorted by FACS using an FITC-labeled anti-human CD14 antibody (clone MY4-FITC, Coulter Corporation, USA); T lymphocytes were sorted using phytoerythrin-labeled anti-human CD2 antibody (clone 39C1.5-PE, Immunotech, France). Total DNA was extracted from total PBMC and the sorted cells by lysis in a 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate (SDS) solution containing 500 g/ml proteinase K (Boehringer Mannheim). After overnight digest at 50⬚C, the DNA was extracted twice with phenol-chloroform, precipitated, and resuspended in 10 mM Tris, 1 mM EDTA (pH 8.0). In other tissue samples, genomic DNA was extracted using the same procedure as for PBMC, except the lysis step which was carried out in a 8 M urea, 10 mM EDTA, 300 mM NaCl, 10 mM Tris, 1% SDS buffer containing 500 mg proteinase K/ml overnight at 37⬚C. PCR analysis was performed using 750 ng DNA as described above. A control reaction was performed to detect cytochrome B sequence using two primers (5⬘-CCCCTCAGAATGATATTTGTCCTCA-3⬘/5⬘-CCATCCAACATCTCAGCATGATGAAA-3⬘) which amplified a 350-bp fragment.
ACKNOWLEDGMENTS We thank Jean-Michel Heard (Pasteur Institute, Paris) and Nick Muzyczka (University of Florida, Gainesville) for critically reading the manuscript and helpful discussions. This work was supported by the Association Française contre les Myopathies (AFM), Vaincre les Maladies Lysosomales (VML), the Association Nantaise de Thérapie Génique (ANTG), and the Fondation pour la Thérapie Génique en Pays de la Loire. D.F. was supported by a fellowship from the Ministère de la Recherche. RECEIVED FOR PUBLICATION MAY 31; ACCEPTED SEPTEMBER 28, 2001.
REFERENCES 1. Muzyczka, N. (1992). Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr. Top. Microbiol. Immunol. 158: 97–129. 2. Berns, K. I., and Giraud, C. (1996). Biology of adeno-associated virus. Curr. Top. Microbiol. Immunol. 218: 1–23. 3. Snyder, R. O. (1999). Adeno-associated virus-mediated gene delivery. J. Gene Med. 1: 166–175. 4. Herzog, R. W., et al. (1997). Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc. Natl. Acad. Sci. USA 94: 5804–5809. 5. Herzog, R. W., et al. (1999). Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat. Med. 5: 56–63. 6. Kay, M. A., et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24: 257–261. 7. Xiao, X., et al. (2000). Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy. J. Virol. 74: 1436–1442. 8. Bohl, D., Salvetti, A., Moullier, P., and Heard, J. M. (1998). Control of erythropoietin delivery by doxycyclin in mice after intramuscular injection of adeno-associated vector. Blood 92: 1512–1517. 9. Bohl, D., Bosch, A., Cardona, A., Salvetti, A., and Heard, J. M. (2000). Improvement of erythropoiesis in -thalassemic mice by continuous erythropoietin delivery from muscle. Blood 95: 2793–2798.
566
10. Song, S., et al. (1998). Sustained secretion of human ␣-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc. Natl. Acad. Sci. USA 95: 14384–14388. 11. Simon, R. H., et al. (1993). Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study. Hum. Gene Ther. 4: 771–780. 12. Sene, C., et al. (1995). Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates. Hum. Gene Ther. 6: 1587–1593. 13. Nunes, F. A., Furth, E. E., Wilson, J. M., and Raper, S. E. (1999). Gene transfer into the liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration. Hum. Gene Ther. 10: 2515–2526. 14. Crystal, R. G., et al. (1994). Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat. Genet. 8: 42–51. 15. Bellon, G., et al. (1997). Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial. Hum. Gene Ther. 8: 15–25. 16. Gossen, M., and Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracyclin-responsive promoters. Proc. Natl. Acad. Sci. USA 89: 5547–5551. 17. Salvetti, A., et al. (1998). Factors influencing recombinant adeno-associated virus production. Hum. Gene Ther. 9: 695–706. 18. Grimm, D., Kern, A., Rittner, K., and Kleinschmidt, J. (1998). Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum. Gene Ther. 9: 2745–2760. 19. Zolotukhin, S., et al. (1999). Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6: 973–985. 20. Pruchnic, R., et al. (2000). The use of adeno-associated virus to circumvent the maturation-dependent viral transduction of muscle fibers. Hum. Gene Ther. 11: 521–536. 21. Grossman, Z., et al. (1992). Detection of adeno-associated virus type 2 in human peripheral blood cells. J. Gen. Virol. 73: 961–966. 22. Hernandez, Y. J., et al. (1999). Latent adeno-associated virus infection elicits humoral but not cell-mediated immune responses in a nonhuman primate model. J. Virol. 73: 8549–8558. 23. Afione, S. A., et al. (1996). In vivo model of adeno-associated virus vector persistence and rescue. J. Virol. 70: 3235–3241. 24. Conrad, C. K., et al. (1996). Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther. 3: 658–668. 25. Monahan, P. E., et al. (1998). Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia. Gene Ther. 5: 40–49. 26. Snyder, R. O., et al. (1999). Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat. Med. 5: 64–70. 27. Soochuen, T. K., Pettis, R. M., Mhatre, A. N., and Lalwani, A. K. (2000). Safety of adenoassociated virus as cochlear gene transfer vector: analysis of distant spread beyond injected cochleae. Mol. Ther. 2: 368–373, doi:10.1006/mthe.2000.0129 28. Nathwani, A. C., et al. (2001). Factors influencing in vivo transduction by recombinant adeno-associated viral vectors expressing the human factor IX cDNA. Blood 97: 1258–1265. 29. Donsante, A., et al. (2001). Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors. Gene Ther. 8: 1343–1346. 30. Dranoff, G., et al. (1993). Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long lasting anti-tumor immunity. Proc. Natl. Acad. Sci. USA 90: 3539–3543. 31. Samulski, R. J., Chang, L. S., and Shenk, T. (1989). Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol. 63: 3822–3828. 32. Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193–199. 33. Favre, D., et al. (2000). Hyaluronidase enhances recombinant adeno-associated virus (rAAV)-mediated gene transfer in the rat skeletal muscle. Gene Ther. 7: 1417–1420.
MOLECULAR THERAPY Vol. 4, No. 6, December 2001 Copyright © The American Society of Gene Therapy