RESUMO
Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.
Assuntos
Antígenos CD/genética , Terapia Genética/métodos , Glicoproteínas/genética , Células-Tronco Hematopoéticas/imunologia , Lentivirus/genética , Peptídeos/genética , Antígeno AC133 , Animais , Antígenos CD/imunologia , Antígenos CD34/genética , Antígenos CD34/imunologia , Expressão Gênica , Vetores Genéticos , Glicoproteínas/imunologia , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/patologia , Doença Granulomatosa Crônica/terapia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Peptídeos/imunologia , Cultura Primária de Células , Transdução Genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy. We describe here a novel vector (CD8-LV) derived from lentivirus, which delivers genes exclusively and specifically to CD8(+) cells. CD8-LV mediated stable in vitro and in vivo reporter gene transfer as well as efficient transfer of genes encoding TCRs recognizing the melanoma antigen tyrosinase. Strikingly, T cells genetically modified with CD8-LV killed melanoma cells reproducibly more efficiently than CD8(+) cells transduced with a conventional lentiviral vector. Neither TCR expression levels, nor the rate of activation-induced death of transduced cells differed between both vector types. Instead, CD8-LV transduced cells showed increased granzyme B and perforin levels as well as an up-regulation of CD8 surface expression in a small subpopulation of cells. Thus, a possible mechanism for CD8-LV enhanced tumor cell killing may be based on activation of the effector functions of CD8(+) T cells by the vector particle displaying OKT8-derived CD8-scFv and an increase of the surface density of CD8, which functions as coreceptor for tumor-cell recognition. CD8-LV represents a powerful novel vector for TCR gene therapy and other applications in immunotherapy and basic research requiring CD8(+) cell-specific gene delivery.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica/genética , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/fisiologia , Células Cultivadas , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HEK293 , Humanos , Imunoterapia Adotiva/métodos , Células Jurkat , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Neoplasias/genética , Especificidade de Órgãos/genética , Regulação para Cima/genética , Regulação para Cima/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.
Assuntos
Células Endoteliais/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Neurônios/metabolismo , Antígeno AC133 , Animais , Antígenos CD/genética , Antígenos CD20/genética , Células Cultivadas , Glicoproteínas/genética , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Receptores de AMPA/genéticaRESUMO
We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Vírus do Sarampo/metabolismo , Receptor ErbB-2/metabolismo , Anticorpos de Cadeia Única/metabolismo , Proteínas Virais de Fusão/metabolismo , Linhagem Celular , Citometria de Fluxo , Humanos , Immunoblotting , Receptor ErbB-2/genética , Anticorpos de Cadeia Única/genética , Proteínas Virais de Fusão/genéticaRESUMO
Hematopoietic stem cells (HSCs) are an important target cell population for gene therapy since they can reconstitute the entire hematopoietic system. HSC-enriched cell populations can be recognized based on cell surface marker expression, such as CD34, which is broadly expressed on immature and partially differentiated cells. In mice, co-expression of CD34 and CD105 was previously shown to be relatively more specific for the most immature, long-term repopulating HSCs. Here, we evaluated whether CD105, which is expressed on 30%-80% of CD34(+) cells, is a marker also for human long-term repopulating HSCs. Therefore, we tracked the mature progeny of CD34(+) cells transduced with the CD105-targeted lentiviral vector CD105-LV in xenotolerant mice. Transduction was blocked with soluble CD105 protein confirming specificity. Importantly, CD105-LV transduced human CD34(+) cells engrafted in NOD-scid IL2Rγ(-/-) mice with up to 20% reporter gene-positive cells detected long term in all human hematopoietic lineages in bone marrow (BM), spleen, and blood. In addition, competitive repopulation experiments in mice showed a superior engraftment of CD105-LV transduced CD34(+) cells in BM and spleen compared with cells transduced with a conventional nontargeted lentiviral vector. Thus, human CD34(+)/CD105(+) cells are enriched for early HSCs with high repopulating capacity. Targeting this cell population with CD105-LV offers a novel gene transfer strategy to reach high engraftment rates of transduced cells and highlights the applicability of receptor-targeted vectors to trace cell subsets offering an alternative to prospective isolation by surface markers.
Assuntos
Antígenos CD/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Superfície Celular/metabolismo , Transplante de Células-Tronco/métodos , Animais , Antígenos CD/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Diferenciação Celular , Endoglina , Marcação de Genes , Células-Tronco Hematopoéticas/citologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Superfície Celular/genéticaRESUMO
B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction.
Assuntos
Antígenos CD19/metabolismo , Antígenos CD20/metabolismo , Linfócitos B/metabolismo , Vetores Genéticos , Ativação Linfocitária , Vírus do Sarampo , Transdução Genética , Antígenos CD19/genética , Antígenos CD20/genética , Linfócitos B/citologia , Fase G1/genética , Células HEK293 , HumanosRESUMO
Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of α-MV antibody-positive human plasma. At plasma dilution 1:160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1:80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans.
Assuntos
Lentivirus/genética , Vírus do Sarampo/genética , Transdução Genética , Proteínas Virais de Fusão/genética , Internalização do Vírus , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hemaglutininas Virais/genética , Humanos , Lentivirus/imunologia , Lentivirus/fisiologia , Vírus do Sarampo/imunologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.