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Receptor-Mediated Gene Delivery to Human Peripheral Blood Mononuclear Cells Using Anti-CD3 Antibody Coupled to Polyethylenimine

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Receptor-Mediated Gene Delivery to Human Peripheral Blood Mononuclear Cells Using Anti-CD3 Antibody Coupled to Polyethylenimine Gene Therapy (2001) 8, 362–368 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Receptor-mediated gene delivery to human peripheral blood mononuclear cells using anti-CD3 antibody coupled to polyethylenimine MM O’Neill, CA Kennedy, RW Barton and RJ Tatake Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA Gene transfer to primary cells, especially to lymphoid cells, expression was detected until 96 h with a gradual diminution using a nonviral delivery system has been very challenging. in the signal after 48 h. Receptor-mediated gene delivery In the present studies, we have used a cationic polymer, was successfully used for freshly isolated, as well as pre- polyethylenimine (PEI) coupled to an anti-CD3 antibody for viously frozen lymphocyte samples. The transfections per- achieving receptor-mediated gene delivery to human periph- formed using ligands other than anti-CD3 were not as eral blood mononuclear cells (PBMC). Naive, unstimulated efficient as anti-CD3-PEI. These results suggest that in PBMC did not express transfected genes, whereas the addition to receptor-mediated endocytosis, signaling sub- transgenes were expressed efficiently in PHA activated sequent to engagement of the CD3 receptor with anti-CD3- PBMC. Transiently expressed gene products were detected PEI appears to be important for the efficacy of anti-CD3-PEI maximally at 24 and 48 h following transfection. Gene mediated gene delivery. Gene Therapy (2001) 8, 362–368. Keywords: gene delivery; nonviral; receptor-mediated; lymphocytes Introduction providing a co-stimulatory signal has increased the efficiency of retroviral transduction of lymphoid cells.9 Peripheral blood mononuclear cells (PBMC), consist of Pseudotyped retroviruses have also been used in order subpopulations of cells that are involved in specialized to increase the efficiency to transduce lymphoid cells.10–13 effector and regulatory functions in response to immuno- In these studies envelope protein of Moloney murine leu- logical stimuli. As a general rule, T lymphocytes are kemia virus has been replaced by the G glycoprotein of responsible for cellular immune response and humoral vesicular stomatitis virus to achieve vast host range,10,11 response is mediated by B lymphocytes. Due to their or with the glycoproteins of HIV for targeting specific, immunological specificity, genetically modified effector CD4-positive T cells.12,13 In comparison to retroviruses, 1 cells have been used for developing cell-based therapies. adenoviral vectors are used less frequently for transduc- Genetically modified lymphoid cells have been used to ing lymphoid cells,14,15 partly because lymphoid cells deliver therapeutic proteins in experimental rheumatoid inadequately express the CAR (receptor for adenovirus 2 3 arthritis, adenosine deaminase deficiency, and graft- and coxsackie virus) receptors that are essential for the 4,5 versus-host disease. Genetically modified immune cells attachment and entry of adenoviruses into cells.15 Besides 6 have also been shown to increase antitumor reactivity. retro- and adenoviral vectors, adeno-associated,16 and In addition to their potential for developing cell-based SV40-based17 viral vectors have been used in limited therapies, transiently transfected lymphoid cells are of studies to transduce lymphoid cells. Although the viral value to understand the role of certain genes that may be vectors continue to evolve to offer a better system, the differentially expressed in different lymphoid subpopula- major disadvantages are due to limitation of the length 7,8 tions in response to a stimulus. For this purpose, PBMC of DNA to be inserted, difficulties in consistent pro- provide an easily accessible cell population, which more duction of vectors and immunogenicity of viral proteins. closely represents a physiological system than established Nonviral gene delivery systems, on the other hand, cell lines. Unfortunately, PBMC have proven to be very appear to allow more flexibility with respect to the DNA difficult to transfect, using both viral as well as nonviral sequences to be inserted into cells but provide much gene delivery systems. lower efficiency of transfection. The attempts to facilitate Among viral vectors, retroviral vectors are commonly transfer of DNA into primary T or B lymphocytes using used for gene delivery to lymphoid cells because of their nonviral vectors have met with limited success.18,19 Sev- ability to transduce hematopoietic cells, although they eral rate-limiting steps, such as suboptimal attachment generally offer low transduction efficiency. In some cases, and internalization through the cell membrane, and/or inadequate release of vector–DNA complexes from the endosomes and transport of DNA to the nucleus, may be Correspondence: RJ Tatake partially responsible for the inefficiency of nonviral vec- Received 28 February 2000; accepted 30 November 2000 tors for gene delivery to lymphoid cells. To overcome Nonviral gene delivery to lymphocytes MM O’Neill et al 363 some of these hurdles, nonviral vectors have been modi- fied by covalent attachment of various ligands, which facilitate entry of DNA into cells via specific receptors.20–23 This approach was explored by Buschle et al24 to transfect primary lymphocytes using an anti-CD3 antibody conju- gated to poly-l-lysine to facilitate gene transfer via the CD3 receptor. Using anti-CD3-poly-l-lysine for gene transfer, they observed very efficient gene delivery to T lymphoblastoid cell lines, however, the efficiency of gene expression in prestimulated PBMC was low. Recently, Kircheis et al25 used a cationic polymer, poly- ethylenimine (PEI), to investigate combined effect of specificity of receptor-mediated gene delivery and high- transfection potential of PEI. PEI has been shown to be an efficient nonviral vector for gene delivery to a variety of cell types, both in vitro, as well as in vivo.26–32 The dis- Figure 1 Luciferase activity in human PBMC transfected with pGL3-con- tinctive properties of PEI, such as DNA-binding and con- trol DNA. Human PBMC were transfected with pGL3 control vector densation, along with its high buffering capacity is con- using electroporation (a) or anti-CD3-PEI (b). PBMC were prestimulated sidered to protect DNA from degradation.29 PEI acts as with PHA (1 ␮g/ml) for 48 h and supplemented with hrIL-2 (10 U/ml) a ‘proton sponge’ in the acidic environment of the lyso- for 24 h before transfection. Twenty-four hours after transfection, each transfected culture was split into two cultures. One culture was stimu- somes, and promote release of DNA from the lyso- + somes.33 Furthermore, PEI is also shown to promote lated with PMA (20 ng/ml) for 20 h (black bars, ), and the other was left unstimulated (gray bars, −). Cells were harvested and luciferase transgene localization to the nucleus in mammalian activity was determined as described in Materials and methods. The values 34,35 cells. When these properties of PEI were combined for relative light units (RLU) for the luciferase expression are given above with the specific mechanism of receptor-mediated gene each histogram since error bars are not visible due to relatively small delivery, ligand-conjugated PEI resulted in higher trans- standard deviation and the logarithmic scale. fection efficiency in various tumor cell lines.25 In the stud- ies reported here, we have used PEI coupled to various ligands specific for the receptors present on PBMC to was further augmented approximately 60% when trans- assess gene delivery to human PBMC. It was observed fected cells were treated with PMA (from RLU 45 406 ± that PBMC were efficiently transfected by anti-CD3-PEI, 851 to 72 361 ± 378). whereas anti-ICAM1-PEI and anti-MHC class I-PEI did Similar to various nonviral gene delivery vectors, non- not transfer DNA effectively. The results suggest that in targeted PEI was not effective in transfecting PBMC (data addition to ligand–receptor interaction and endocytosis not shown). Based on the specificity of anti-CD3-PEI to of the DNA/vector complexes, efficiency of expression target CD3-positive cells in the studies by Kirches et al,25 of a transgene is affected by signaling events following it is highly likely that the cell population expressing the receptor engagement. luciferase may be T lymphocytes. However, since PBMC were not fractionated to enrich T lymphocytes, the trans- fected cell population has been referred to as PBMC. The Results efficiency of transfection, in terms of the transgene expression, was variable in different PBMC samples. The Transfection of human PBMC with pGL3 plasmid DNA average luciferase expression without PMA stimulation Expression of the luciferase gene in human PBMC was of PBMC from nine different donors was 14 848 RLU/1 assessed following transfection of PBMC with pGL3 vec- × 105 cells/s (range 1236–45 406 RLU/1 × 105 cells/s). tor using various nonviral gene delivery carriers. The Stimulation of the transfected cells with PMA did not attempts to transfect PBMC using Superfect, Lipofectam- affect the variability in gene expression. Despite the ine or DEAE-dextran were unsuccessful (data not variability, luciferase activity was consistently seen in all shown). However, consistent gene expression was achi- PBMC samples transfected using anti-CD3-PEI. eved when PBMC were transfected either by electropor- ation or receptor-mediated gene delivery using anti-CD3- Effect of prestimulation of PBMC on the efficiency of PEI conjugates. The results from a representative
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