Leukemia & Lymphoma, March 2011; 52(3): 483–490
ORIGINAL ARTICLE: RESEARCH
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
Efficient gene transfer with pseudotyped recombinant adeno-associated viral vectors into human chronic myelogenous leukemia cells
¨ RGEN A. KLEINSCHMIDT4, LEOPOLD SELLNER1,2*, MARLON R. VELDWIJK1,3*, JU STEPHANIE LAUFS5,6, JULIAN TOPALY7, STEFAN FRUEHAUF7, W. JENS ZELLER1, & FREDERIK WENZ3 1
Pharmacology of Cancer Treatment, German Cancer Research Center, Heidelberg, Germany, 2Department of Internal Medicine V, University of Heidelberg, Heidelberg, Germany, 3Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany, 4Applied Tumor Virology, German Cancer Research Center, Heidelberg, Germany, 5Department of Experimental Surgery, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany, 6Molecular Oncology of Solid Tumors, German Cancer Research Center, Heidelberg, Germany, and 7 Center for Tumor Diagnostics and Therapy, Paracelsus-Klinik, Osnabru¨ck, Germany (Received 13 April 2010; accepted 17 August 2010)
Abstract Gene transfer into chronic myelogenous leukemia (CML) cells may become of relevance for overcoming therapy resistance. Single-stranded pseudotyped adeno-associated viruses of serotypes 2/1 to 2/6 (ssAAV2/1–ssAAV2/6) were screened on human CML cell lines and primary cells to determine gene transfer efficiency. Additionally, double-stranded selfcomplementary vectors (dsAAVs) were used to determine possible second-strand synthesis limitations. On human CML cell lines, ssAAV2/2 and ssAAV2/6 were most efficient. On primary cells, ssAAV2/6 proved significantly more efficient (4.1 + 2.5% GFPþ cells, p ¼ 0.011) than the other vectors (51%). The transduction efficiency could be significantly increased (45.5 + 13.4%) by using dsAAV2/6 vectors (p 5 0.001 vs. ssAAV2/6). In these settings, our data suggest conversion of single- to double-stranded DNA and cell binding/entry as rate-limiting steps. Furthermore, gene transfer was observed in both late and earlier CML (progenitor) populations. For the first time, efficient AAV gene transfer into human CML cells could be shown, with the potential for future clinical application.
Keywords: Adeno-associated virus, chronic myelogenous leukemia, gene transfer, pseudotyped vector
Introduction Among different types of leukemia, chronic myelogenous leukemia (CML) is of high relevance. CML was the first malignancy for which a special chromosomal abnormality could be directly linked to the cause of the disease. Due to the translocation of the Philadelphia chromosome [Ph; t(9;22)], a fusion gene (BCR–ABL) [1] is generated, resulting in the production of a constitutively active cytoplasmic tyrosine kinase that is responsible for the onset of CML. The BCR-ABL [2] inhibitor imatinib mesylate is highly effective, well tolerated and represents the
current standard of treatment in CML. However, it does not provide a cure for CML in most patients and emergence of imatinib-resistant CML cell clones may represent a serious clinical challenge [3–5]. In order to overcome this problem, novel therapeutic options are imperative. Gene therapy vectors containing either suicide [6] and/or immune-stimulating genes [7,8] may become promising tools against this disease. For clinical gene therapeutic approaches, an efficient and safe vector system is required. In this study, vectors based on the small (20 nm) non-enveloped, single-stranded DNA-containing parvovirus adenoassociated virus (AAV) were used. Recombinant
Correspondence: Dr. M. R. Veldwijk, Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, Theodor-KutzerUfer 1-3, D-68135 Mannheim, Germany. Tel: þ49-621-3833750. Fax: þ49-621-3833493. E-mail:
[email protected] *Contributed equally ISSN 1042-8194 print/ISSN 1029-2403 online Ó 2011 Informa UK, Ltd. DOI: 10.3109/10428194.2010.545460
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
484
L. Sellner et al.
AAV-based vectors have an advantageous safety profile due to their low immunogenicity [9], the lack of any associated pathogenicity [10,11], and their primarily episomal localization [12–15]. Singlestranded recombinant AAV (ssAAV) vectors, especially those based on serotype 2 (ssAAV2), have been extensively used in preclinical and clinical trials, including for instance the treatment of clotting factor disorders [16], cystic fibrosis [17], and several types of cancer [18,19]. However, standard ssAAV2 vectors lacked the required transduction efficiency on leukemia cell lines [20,21], as well as on primary human CML cells [21]. Recently, we demonstrated [20,21] that vector modification using an AAV random peptide display [22] can increase transduction efficiency on leukemia cell lines as well as on primary human CML cells, even though the results obtained on primary human CML cells were deflating, with gene expression efficiencies of less than 5% [21]. It has been demonstrated that several serotypes differ in gene transfer efficiency due to their tissue tropism [23]. Therefore, the use of pseudotyped ssAAV2 vectors (designated ssAAV2/x: containing the vector genome of serotype 2 and capsid of serotype x) may also lead to differences in tropism, thereby possibly resulting in increased transduction efficiency on CML cells, and these vectors were screened in the present study. We have already been able to show promising results with pseudotyped AAV vectors on primary CD34þ peripheral blood progenitor cells (PBPCs) [24]. A further potentially rate-limiting step for effective gene transfer, besides cell attachment and entry, is the requirement of second-strand synthesis from the single-stranded AAV genome before transgene expression [25]. To overcome this potential obstacle, double-stranded, self-complementary AAV (dsAAV) vectors [26,27], thereby potentially allowing higher expression rates, were additionally tested. In this investigation we aimed to find the most efficient pseudotyped ssAAV vectors for transduction of human CML cell lines, as well as for primary human CML cells, and to obtain a vector system efficient enough for potential future clinical application. To avoid limitations in transduction efficiency
due to the requirement of second-strand synthesis of the single-stranded AAV genome and to furthermore increase transduction efficiency, the potential of the most efficient dsAAV vectors was also investigated on primary human CML cells.
Materials and methods Cells and cell culture The embryonic kidney cell line 293T was provided by Dr. Kleinschmidt (DKFZ, Heidelberg, Germany) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 5 mg/mL penicillin/streptomycin (media and supplements from Gibco Invitrogen Corp., Karlsruhe, Germany). The BCR–ABL-positive human CML cell lines BV173, EM3, K562, and LAMA84-S were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Imatinib-resistant LAMA84-R cells were kindly provided by the laboratory of Dr. J. Melo (London, UK) [28], and BV173 (CML lymphoid blast crisis), EM3 (CML blast crisis relapse), K562 (CML blast crisis), LAMA84-R (CML blast crisis), and LAMA84-S (CML blast crisis) cells were cultivated in Roswell Park Memorial Institute medium (RPMI; Gibco Invitrogen Corp.) with the same supplements as used for the DMEM medium. LAMA84-R cells were cultured in RPMI additionally supplemented with 1 mM imatinib. Primary human CML samples were provided by the Department of Internal Medicine V (Heidelberg, Germany) and the Center for Tumor Diagnostics and Therapy, Paracelsus-Klinik (Osnabru¨ck, Germany), and obtained from four patients with CML (Table I). These samples contained 226. 600 + 64. 211 leukocytes/mL. Primary human CML cells were grown in Iscove’s modified Dulbecco’s medium (IMDM; Gibco Invitrogen Corp.) with the same supplements as used for the DMEM medium but additionally supplemented with 20 ng/mL thrombopoietin (TPO), 100 ng/mL Flt-3 ligand (FLt3L), and 100 ng/mL stem cell factor (SCF; SCF from
Table I. Patient characteristics. Patient ID
Sex
Age at harvest (years)
Diagnosis
Source
CML 1 CML 2 CML 3 CML 4 Summary
F M M F 2 6 M/2 6 F
69 42 18 31 40 + 22
CML BC, IR CML CP CML CP CML CP 1 6 BC/3 6 CP
PB BM PB PB 1 6 BM, 3 6 PB
CML, chronic myelogenous leukemia; M, male; F, female; BC, blast crisis; IR, imatinib-resistant; CP, chronic phase; PB, peripheral blood; BM, bone marrow.
Gene transfer into leukemia cells Genzyme, Cambridge, MA, USA; TPO and FLt3L from R&D Systems, Minneapolis, MN, USA). The investigation on viral gene transfer was approved by the Ethical Committee of the Medical Faculty of the University of Heidelberg and informed consent was obtained from each patient.
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
Production and titration of AAV particles For pseudotyped ssAAV production, the plasmids MRV-Ef1a-eGFP (ssAAV2 vector) [19] and DP1DP6 (pseudotyping AAV helper) [23] were used. For the dsAAV stocks, plasmid MRV-Ef1a-eGFP was replaced by ds-CMV-GFP [27]. All vectors were produced and titrated as described previously [20]. Transduction In suspension growing human CML cell lines were seeded on the day of transduction into 24-well plates at a density of 2 6 104 cells/well in 300 mL of the respective medium, and primary human CML cells were seeded at a density of 4 6 104 cells/well with 500 mL of IMDM medium supplemented with SCF, TPO, and FLt3L as described above. In all experiments, cells were transduced with 100 transducing units/cell of the respective vectors, incubated for 72 h, thereafter harvested, and analyzed by flow cytometry, as described previously [20]. Fluorescence activated cell sorting analysis For acquisition and analysis, a FACSCalibur flow cytometer (Becton-Dickinson GmbH, Heidelberg, Germany) equipped with an air-cooled 488 nm argon laser was used. Data were processed using Cellquest software (Becton-Dickinson). Before acquisition, propidium iodide (10 mg/mL; Sigma) was added to samples, to exclude dead cells from analysis. Ten thousand events were acquired. Green fluorescent protein (GFP) was measured on the Fl1-channel and plotted against side-scatter as described previously [29]. GFP expression was measured against uninfected control cells, thereby correcting the auto- and nonspecific fluorescence. The value of GFP expression is given as the percentage of GFP-positive cells. For analysis of the gene transfer distribution among the CD34 and CD38 populations, additionally the human anti-CD34-phycoerythrin (PE) and human antiCD38-peridinin-chlorophyll protein (PerCP) monoclonal antibodies were used (both Becton-Dickinson). Statistics Data are given as mean value + standard deviation (cell line experiments) or mean value + standard
485
error of the mean (primary CML experiments). For each vector, cells from at least three patients per experiment were used; for each patient sample/vector combination, at least three independent experiments were performed. Significance and correlation levels were determined using the one-sided analysis of variance (ANOVA) test with the Tukey post hoc test or the (un)paired, two-sided Student t-test.
Results Transduction efficiency of pseudotyped ssAAV vectors on human CML cell lines To determine gene transfer efficiency of pseudotyped ssAAV vectors, five human CML cell lines (BV173, EM3, K562, LAMA84-S [imatinib-sensitive], and LAMA84-R [imatinib-resistant]) were transduced with ssAAV particles of the pseudotyped serotypes 2/1 to 2/6 (Figure 1). A multiplicity of infection (MOI) of 100 infectious units was used (n ¼ 3). With the exception of LAMA84-S and EM3, ssAAV2/2 was the most efficient on the human CML cell lines: BV173 (22.9 + 3.8%), K562 (72.1 + 6.2%), and LAMA84-R (83.9 + 2.7%), followed by ssAAV2/6. On the human CML cell line EM3, ssAAV2/6 was the only vector showing significant gene transfer (7.5 + 1.2%). Transduction of BV173 and K562 cells with ssAAV2/1 vectors also resulted in solid expression of the transgene (7.2 + 1.3% and 35.1 + 8.0%, respectively). Interesting observations were made on the human CML cell line LAMA84; here an obvious difference in susceptibility for AAV vectors between the imatinib-sensitive cell line LAMA84-S and the imatinib-resistant counterpart LAMA84-R could be observed. On LAMA84-S cells, ssAAV2/6 (22.5 + 4.6%) was significantly more efficient compared to all other pseudotyped vectors (p 5 0.001), with lower gene transfer levels for ssAAV2/1 (6.2 + 1.7%), ssAAV2/2 (2.5 + 1.2%), and ssAAV2/4 (3.6 + 1.2%) vectors (ssAAV2/3 and ssAAV2/5, 51%). Although ssAAV2/6 transgene expression remained similar on the LAMA84-R cells (26.0 + 1.3%), ssAAV2/2 became the most efficient vector (83.9 + 2.7%) followed by ssAAV2/1 (71.8 + 4.5%) and ssAAV2/5 (4.6 + 0.8%). Using ssAAV2/4 vectors, low-level gene transfer into LAMA84-R cells (1.3 + 0.4%) was observed. Pseudotyped ssAAV-mediated gene transfer into primary human CML cells To show proof-of-principle, gene transfer efficiency of pseudotyped ssAAV 2/1 to 2/6 vectors (MOI 100) was determined on primary human CML cells (for
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
486
L. Sellner et al.
Figure 1. Gene transfer efficiency of pseudotyped ssAAV vectors on human CML cell lines. A multiplicity of infection of 100 was used. For ssAAV2/1, ssAAV2/2, and ssAAV2/6 significant gene transfer could be detected.
each, n 3; Figure 2). On these cells, only ssAAV2/6 (4.1 + 2.5%) vectors could show significant gene transfer efficiency compared to all other pseudotyped vectors (p ¼ 0.011); for all other vectors (ssAAV2/1 to ssAAV2/5) 1% gene transfer was observed. Of note, a high inter-patient variability was observed. Comparison of transduction efficiency between singlestranded and double-stranded AAV vectors on primary human CML cells Although significant gene transfer could be observed after transduction of primary human CML cells with pseudotyped ssAAV vectors, these were moderate. In order to potentially increase the gene transfer efficiency and determine a potential block in singleto double-strand synthesis, double-stranded selfcomplementary pseudotyped vectors (dsAAV2/1, dsAAV2/2, and dsAAV2/6) were tested on primary human CML cells (n 3; MOI 100; Figure 3). All tested self-complementary vectors were significantly more efficient than their single-stranded counterparts (dsAAV2/1: 11.4 + 6.5%, p 5 0.05; dsAAV2/2: 15.6 + 6.7%, p 5 0.001; dsAAV2/6: 45.5 + 13.4%, p 5 0.001) (Figure 3), thereby suggesting high relevance of a block in single- to second-strand synthesis in primary human CML cells. The highest gene transfer efficiency was observed using dsAAV2/ 6 (up to 70% GFP-positive cells). As observed with the pseudotyped ssAAV vectors, a high inter-patient variability was obtained, yet as expected, a significant correlation between the susceptibility of a patient’s CML cells to pseudotyped ssAAV and dsAAV vectors was observed (r ¼ 0.47, p ¼ 0.012). The latter suggests that the susceptibility of primary human
Figure 2. Gene transfer efficiency of pseudotyped ssAAV vectors on primary human CML cells. At a multiplicity of infection of 100, ssAAV2/6 vectors were significantly more efficient than all other tested vectors (p ¼ 0.011). Data are given as mean + standard error of the mean.
Figure 3. Comparison of gene transfer efficiency between dsAAV vectors with their single-stranded counterpart on primary human CML cells. At a multiplicity of infection of 100, all tested dsAAV vectors were significantly more efficient compared to their singlestranded counterpart. Data are given as mean + standard error of the mean. *p 5 0.05.
Gene transfer into leukemia cells
vector, for two primary human CML samples the gene transfer distributions among the CD34 and CD38 populations after transduction with a dsAAV2/ 6 vector were obtained (Figure 4). Concerning the CD34 antigen, a progenitor marker, a higher proportion of CD34dull and CD34high (late and early progenitors, respectively) expressed the transgene GFP after transduction compared with the CD347 population. Whereas for the CD347 population only
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
CML cells is primarily dependent on cell binding and entry of the viral particles. Of note, similar to the high gene transfer rates in the imatinib-resistant cell line LAMA84-R, also for primary CML cells from an imatinib-resistant patient, solid gene transfer could be observed for both ss- and dsAAV vectors (ssAAV2/6: 8.5 + 6.4%; dsAAV2/6: 64.9 + 4.1%). To determine which subpopulation within the primary human CML cells was targeted by the
487
Figure 4. Gene transfer distribution among the CD34 and CD38 populations after transduction of primary human CML cells from two patients with a dsAAV2/6 vector (multiplicity of infection of 20). For the CD34 antigen, proportionally more CD34dull and CD34high cells were positive for the transgene GFP than in the CD347 population. Although for the CD38 antigen almost no CD387 cells were observed in both CML samples, gene transfer was homogeneously distributed among the CD38dull and CD38high populations.
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
488
L. Sellner et al.
one in 17.1 + 1.4 cells was GFPþ, in the CD34þ population this ratio was readily larger (one in 6.2 + 0.8 cells). This suggests an efficient gene transfer using AAV vectors in both the primitive and more differentiated CML populations. Of note, a reduced MOI (20) was used to reduce the probability of gene transfer of multiple virus particles in one cell and thereby ensure a sound distribution of all parameters in fluorescence activated cell sorting (FACS) plots. For the CD38 antigen, a marker for progenitor maturity (less CD38 expression correlates with a more primitive phenotype), almost no CD387 cells were observed in the screened primary CML samples (50.1%). Gene transfer among the CD38dull and CD38high populations was homogeneously distributed, thereby suggesting no preference. Discussion In this study, the susceptibility of human CML cell lines and primary human CML cells for pseudotyped AAV vectors was determined with the goal of obtaining an efficient AAV vector for the transduction of primary human CML cells and thereby showing proof-of-principle. Therefore, human CML cell lines were transduced with pseudotyped ssAAV vectors and their gene transfer efficiency determined. Although differences in their susceptibility to the vectors were observed, overall ssAAV2/1, ssAAV2/2, and ssAAV2/6 showed the highest gene transfer in these cells. As observed in earlier experiments with normal ssAAV2 and some ssAAV2-based capsid mutant vectors on the imatinib-sensitive CML cell line LAMA84-S and its imatinib-resistant counterpart LAMA84-R [20], also with the pseudotyped ssAAV vectors differences in gene transfer efficiency between both cell lines were observed. The pseudotyped ssAAV2/1 and ssAAV2/2 vectors, being inefficient on LAMA84-S cells (fewer than 8% GFP-positive cells), showed dramatic increases in gene transfer (up to 80%) on the imatinib-resistant LAMA84-R cell line. Of note, both cell lines showed a similar susceptibility toward only the ssAAV2/6 vector (approximately 25% GFPpositive cells). For all other vectors, very low gene transfer rates were observed in both cell lines. These data, especially those for ssAAV2/6, allow discarding of an earlier hypothesis [20] that the increase in gene transfer efficiency in the LAMA84-R cell line observed for ssAAV is caused by an increase in cellular stress due to the cell line being cultured under constant 1 mM imatinib to retain its resistant phenotype. It has been previously shown that cellular stress can increase the gene transfer efficiency of ssAAV vectors [30,31].
After screening the pseudotyped vectors on human CML cell lines, the susceptibility of primary CML cells from patients for these vectors was determined. For the first time, we could show that gene transfer into primary human CML cells was possible with pseudotyped AAV vectors. With single-stranded pseudotyped AAV vectors, ssAAV2/6 proved superior to all other tested pseudotyped ssAAV vectors. Although significant, the obtained gene transfer efficiency was still relatively low (4.1 + 2.5%), and a high variability between the different patient samples was observed. On the other hand, comparing these data to our laboratory’s last gold standard, an ssAAV2-based capsid mutant (1.8 + 0.5% GFPpositive cells) obtained by applying an ssAAV random peptide library on a CML cell line (K562) in earlier experiments [21], primary human CML cells transduced with pseudotyped ssAAV2/6 vectors showed higher gene transfer rates. As shown previously on primary human CD34þ PBPCs [24], we expected the conversion of the single-stranded AAV genome to a doublestranded version as a possible rate-limiting step for efficient gene transfer into primary human CML cells. Therefore the self-complementary, doublestranded vectors dsAAV2/1, dsAAV2/2, and dsAAV2/6 were tested on primary human CML cells, and all three vectors showed significant superiority compared to their single-stranded counterparts (p 5 0.026) with up to 70% GFP-expressing cells. These findings suggest that second-strand synthesis, besides cell binding and entry, seems to be the main limiting step for efficient expression after gene transfer with ssAAV vectors into these target cells. Of note, no significant differences in gene transfer efficiency between human imatinib-resistant and -sensitive primary CML cells were observed for both ss- and dsAAV vectors, thereby offering solid gene transfer in both cell populations. This may become of relevance for potential future clinical applications. A further increase in of gene transfer rates should be possible with the optimization of transduction procedures including the use of higher MOIs and/or multiple transduction regimes, a commonly used procedure in retroviral transduction protocols [32]. Depending on the potential clinical application, it may be of relevance which populations within the primary CML cell pool are targeted by the AAV vectors. As shown in Figure 4, the more primitive late (CD34dull) and especially early CML progenitors (CD34high) can be readily transduced by our dsAAV2/6 vector. Concerning a further marker for ‘stem cellness’ (CD387/dull), a homogeneous distribution in gene transfer among the CD38dull and CD38high populations was observed. Almost no
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
Gene transfer into leukemia cells CD387 cells were observed, possibly caused by either a low amount of CD387 cells in peripheral blood of patients with CML and/or the use of a PElabeled CD38 antibody (stronger signal than the usually applied CD38-fluorescein isothiocyanate [FITC], which was here not possible due to the use of GFP in our vectors). Of note, at the time of transduction, the number of more primitive CML cells (CD34þ/CD387) can be expected to be higher than that shown in Figure 4, as these cells had been in culture for at least 3 days, during which time they most probably lost ‘stem cellness’. Although we could already show the transduction of primitive CD34þ and even CD38dull progenitors, and this topic is outside of the scope of this work, further investigation on the susceptibility of CML progenitor and stem cells should be performed to further elucidate the susceptibility of all CML populations to AAV vectors, as the latter may be of relevance for certain future clinical applications (e.g. leukemia stem cell gene transfer). Advantages of AAV-based vectors over others are the generally rapid transduction of cells by AAV and the lack of requirement for cell division during transduction [30], thereby allowing transduction of dormant CML cells and a minimization of the ex vivo phase in case of an immunotherapeutic approach. The latter should result in reduced exposure to nonphysiologic conditions. Due to the mainly extrachromosomal localization of AAV-based vectors and thereby low integration rates, the system is suited especially for ‘hit and run’ applications requiring short-term expression, such as immunotherapeutic approaches [7]. For both pseudotyped ssAAV and dsAAV vectors, a high degree of inter-patient variability after transduction of primary human CML cells could be detected (Figures 2 and 3). Although outside of the scope of this study, this seems to be primarily caused by differences in vector binding and/or entry efficiency between the different patient samples and less by intracellular trafficking, as a high correlation between the ssAAV and dsAAV vector data was observed. This ought to be further investigated using a larger patient collective, thereby possibly identifying essential factors for the variability, and new strategies could be developed to further increase gene transfer efficiency into primary human CML cells or identify AAV-resistant patients with CML. Of note, although the results for the ssAAV vectors on human CML cell lines roughly correlated with the data for primary human CML cells, caution should be taken when relying only on cell line data, as from our CML cell line results, it was not expected that ssAAV2/6 vectors would be the most efficient in primary cells.
489
Summarizing, we were able to identify AAV-based vectors that for the first time resulted in high gene transfer efficiency into both human CML cell lines and primary human CML cells, thereby showing proof-of-principle and offering a promising system for future gene therapeutic approaches for this clinically relevant disease. Acknowledgements We would like to thank Brigitte Geisler (Department of Internal Medicine V, University of Heidelberg), Bernhard Berkus, Hans-Ju¨rgen Engel, and Sigrid Heil (German Cancer Research Center, Heidelberg, Germany) for their excellent technical support. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References 1. Bartram CR, de Klein A, Hagemeijer A, et al. Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia. Nature 1983;306:277–280. 2. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2:561–566. 3. Branford S, Rudzki Z, Walsh S, et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002;99:3472–3475. 4. Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 2001;293:876–880. 5. Shah NP, Nicoll JM, Nagar B, et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002;2:117–125. 6. Rivas C, Miller AR, Collado M, et al. BCR-ABL-expressing cells transduced with the HSV-tk gene die by apoptosis upon treatment with ganciclovir. Mol Ther 2001;3:642–652. 7. Ling X, Wang Y, Dietrich MF, Andreeff M, Arlinghaus RB. Vaccination with leukemia cells expressing cell-surface-associated GM-CSF blocks leukemia induction in immunocompetent mice. Oncogene 2006;25:4483–4490. 8. Yei S, Bartholomew RM, Pezzoli P, et al. Novel membranebound GM-CSF vaccines for the treatment of cancer: generation and evaluation of mbGM-CSF mouse B16F10 melanoma cell vaccine. Gene Ther 2002;9:1302–1311. 9. McPhee SW, Janson CG, Li C, et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med 2006;8:577–588. 10. Blacklow NR, Hoggan MD, Sereno MS, et al. A seroepidemiologic study of adenovirus-associated virus infection in infants and children. Am J Epidemiol 1971;94:359–366. 11. Conrad CK, Allen SS, Afione SA, et al. Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther 1996;3:658–668.
Leuk Lymphoma Downloaded from informahealthcare.com by 95.117.237.235 on 02/17/11 For personal use only.
490
L. Sellner et al.
12. Duan D, Sharma P, Dudus L, et al. Formation of adenoassociated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene expression. J Virol 1999;73:161–169. 13. Flotte TR. Recent developments in recombinant AAVmediated gene therapy for lung diseases. Curr Gene Ther 2005;5:361–366. 14. McCarty DM, Young SM Jr, Samulski RJ. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet 2004;38:819–845. 15. Miller DG, Trobridge GD, Petek LM, et al. Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. J Virol 2005;79:11434–11442. 16. Sarkar R, Tetreault R, Gao G, et al. Total correction of hemophilia A mice with canine FVIII using an AAV 8 serotype. Blood 2004;103:1253–1260. 17. Flotte TR. Adeno-associated virus-based gene therapy for inherited disorders. Pediatr Res 2005;58:1143–1147. 18. Berlinghoff S, Veldwijk MR, Laufs S, et al. Susceptibility of mesothelioma cell lines to adeno-associated virus 2 vectorbased suicide gene therapy. Lung Cancer 2004;46:179–186. 19. Veldwijk MR, Berlinghoff S, Laufs S, et al. Suicide gene therapy of sarcoma cell lines using recombinant adeno-associated virus 2 vectors. Cancer Gene Ther 2004;11:577–584. 20. Sellner L, Stiefelhagen M, Kleinschmidt JA, et al. Generation of efficient human blood progenitor-targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells. Exp Hematol 2008;36:957–964. 21. Stiefelhagen M, Sellner L, Kleinschmidt JA, et al. Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line. Genet Vaccines Ther 2008;6:12. 22. Muller OJ, Kaul F, Weitzman MD, et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol 2003;21:1040–1046.
23. Grimm D, Kay MA. From virus evolution to vector revolution: use of naturally occurring serotypes of adenoassociated virus (AAV) as novel vectors for human gene therapy. Curr Gene Ther 2003;3:281–304. 24. Veldwijk MR, Sellner L, Stiefelhagen M, et al. Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(þ) peripheral blood progenitor cells. Cytotherapy 2010;12:107–112. 25. Sanlioglu S, Monick MM, Luleci G, Hunninghake GW, Engelhardt JF. Rate limiting steps of AAV transduction and implications for human gene therapy. Curr Gene Ther 2001;1: 137–147. 26. McCarty DM, Fu H, Monahan PE, et al. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther 2003;10:2112–2118. 27. Wang Z, Ma HI, Li J, et al. Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther 2003;10:2105–2111. 28. Mahon FX, Deininger MW, Schultheis B, et al. Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood 2000;96:1070–1079. 29. Veldwijk MR, Fruehauf S, Schiedlmeier B, Kleinschmidt JA, Zeller WJ. Differential expression of a recombinant adenoassociated virus 2 vector in human CD34þ cells and breast cancer cells. Cancer Gene Ther 2000;7:597–604. 30. Alexander IE, Russell DW, Miller AD. DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors. J Virol 1994;68:8282–8287. 31. Qing K, Wang XS, Kube DM, et al. Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression. Proc Natl Acad Sci USA 1997;94:10879–10884. 32. Scherr M, Battmer K, Blomer U, et al. Lentiviral gene transfer into peripheral blood-derived CD34þ NOD/SCID-repopulating cells. Blood 2002;99:709–712.
