RESUMO
Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.
Assuntos
Anafilaxia/metabolismo , Células-Tronco Hematopoéticas/imunologia , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Anafilaxia/imunologia , Animais , Dimerização , Humanos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-kit/agonistas , Proteínas Proto-Oncogênicas c-kit/química , Fator de Células-Tronco/química , Fator de Células-Tronco/genéticaRESUMO
Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1,000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors, with each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions. Targeting efficiency was enhanced by target site transcription, while chromatin accessibility was associated with an increased likelihood of targeting. ChromHMM chromatin states characterizing transcription and enhancers in wild-type K562 cells were also associated with increased AAV-HR efficiency with and without target site transcription, respectively. Furthermore, the amenability of a site to targeting was influenced by the endogenous transcriptional level of intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.
Assuntos
Cromatina/genética , Dependovirus/genética , Marcação de Genes/métodos , Técnicas de Transferência de Genes/estatística & dados numéricos , Vetores Genéticos/genética , Recombinação Homóloga , Transgenes , Vetores Genéticos/administração & dosagem , Humanos , Células K562RESUMO
Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by â¼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to â¼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by â¼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by â¼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.
Assuntos
Sistemas CRISPR-Cas , DNA Helicases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Dependovirus/genética , Edição de Genes , Células-Tronco Hematopoéticas/metabolismo , RecQ Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos , Células HeLa , Células-Tronco Hematopoéticas/citologia , Recombinação Homóloga , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Engenharia de Proteínas/métodos , Receptores Fc/genética , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcitose/fisiologia , Animais , Cães , Escherichia coli , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/genética , Cinética , Células Madin Darby de Rim Canino , Nanopartículas , Peptídeos/genética , Peptídeos/metabolismo , Transcitose/genéticaRESUMO
We report the synthesis and characterization of a series of ionizable lysine-based lipids (ILL), novel lipids containing a lysine headgroup linked to a long-chain dialkylamine through an amide linkage at the lysine α-amine. These ILLs contain two ionizable amines and a carboxylate, and exhibit pH-dependent lipid ionization that varies with lipid structure. The synthetic scheme employed allows for the simple, orthogonal manipulation of lipids. This provides a method for the development of a compositionally diverse library with varying ionizable headgroups, tail structures, and linker regions. A focused library of four ILLs was synthesized to determine the impact of hydrophobic fluidity, lipid net charge, and lipid pK(a) on the biophysical and siRNA transfection characteristics of this new class of lipids. We found that manipulation of lipid structure impacts the protonation behavior, electrostatically driven membrane disruption, and ability to promote siRNA mediated knockdown in vitro. ILL-siRNA liposomal formulations were tested in a murine Factor VII model; however, no significant siRNA-mediated knockdown was observed. These results indicate that ILL may be useful in vitro transfection reagents, but further optimization of this new class of lipids is required to develop an effective in vivo siRNA delivery system.
Assuntos
Lipídeos/química , Lipossomos/química , Lisina/análogos & derivados , RNA Interferente Pequeno/administração & dosagem , Animais , Fator VII/genética , Feminino , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Íons/química , Lipídeos/síntese química , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Metal chelation-ligand interactions, such as occur between nitrilotriacetic acid (NTA)-nickel and multihistidines, enable the noncovalent attachment of histidine-modified proteins to liposomes and other particles. We compared three lipids: a mono-NTA lipid (ca. 10 microM affinity) and two tris-NTA lipid derivatives (ca. 3 nM and 0.2 nM affinity) in their ability to retain two different his(6)-containing proteins on NTA-liposomes in the presence of serum or plasma and after intravenous injection in mice. At nanomolar affinities, the off-rate of a his(6)-ligand is sufficiently long so that his(6)-proteins attached to particle surfaces will remain with the particle for hours; thus, we hypothesized that the increased his(6) affinity of multivalent NTA-modified liposomes would retain his(6)-proteins longer both in vitro and in vivo. For each of the three lipids, we found a robust association and complete activity retention of two his(6)-modified proteins: a far red-fluorescent protein, monomeric Katushka (mKate), and a prodrug-converting enzyme, yeast cytosine deaminase (yCD). Proteins associated more tightly in vitro with tris-NTA liposomes than with mono-NTA liposomes in the presence of refiltered fetal calf serum and mouse plasma. Free yCD exchanged with previously associated mKate for tris-NTA binding sites on the liposome surface. This exchange was due to the exchange of the proteins for NTA occupancy and not due to the exchange of tris-NTA lipid out of the liposome. The amount of yCD on the surface was similar if the proteins were co-associated or if mKate was pre-associated. This exchange confirms that NTA associated proteins are in a dynamic state and can exchange with multihistidine proteins in the biological milieu. There was no difference in circulation time of the protein when it was intravenously administered by itself or attached to any of the NTA-modified liposomes because in vivo the protein was rapidly released from the NTA liposomes. Upon recovery from blood, liposomes containing tris-NTA accumulated a different plasma protein profile than control liposomes, suggesting that Ni-NTA specifically interacts with some plasma proteins. The reason for the rapid protein dissociation from the liposome in vivo is not clear; it could be due to displacement by endogenous histidine-containing proteins or to natural chelators that remove nickel from the NTA. Regardless of the cause, improvements in chelator or ligand design are needed before metal chelation will be capable of retaining histidine-modified proteins on NTA liposomes after in vivo administration.
Assuntos
Histidina/química , Lipídeos/química , Lipossomos/química , Ácido Nitrilotriacético/análogos & derivados , Compostos Organometálicos/química , Proteínas/metabolismo , Animais , Meia-Vida , Histidina/metabolismo , Ligantes , Lipossomos/administração & dosagem , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ácido Nitrilotriacético/química , Ácido Nitrilotriacético/metabolismo , Compostos Organometálicos/metabolismo , Ligação Proteica , Proteínas/químicaRESUMO
Despite early successes using recombinant adeno-associated virus (rAAV) vectors in clinical gene therapy trials, limitations remain making additional advancements a necessity. Some of the challenges include variable levels of pre-existing neutralizing antibodies and poor transduction in specific target tissues and/or diseases. In addition, readministration of an rAAV vector is in general not possible due to the immune response against the capsid. Recombinant adeno-associated virus (AAV) vectors with novel capsids can be isolated in nature or developed through different directed evolution strategies. However, in most cases, the process of AAV selection is not well understood and new strategies are required to define the best parameters to develop more efficient and functional rAAV capsids. Therefore, the use of barcoding for AAV capsid libraries, which can be screened by high-throughput sequencing, provides a powerful tool to track AAV capsid evolution and potentially improve AAV capsid library screens. In this study, we examined how different parameters affect the screen of two different AAV libraries in two human cell types. We uncovered new and unexpected insights in how to maximize the likelihood of obtaining AAV variants with the desired properties. The major findings of the study are the following. (1) Inclusion of helper-virus for AAV replication can selectively propagate variants that can replicate to higher titers, but are not necessarily better at transduction. (2) Competition between AAVs with specific capsids can take place in cells that have been infected with different AAVs. (3) The use of low multiplicity of infections for infection results in more variation between screens and is not optimal at selecting the most desired capsids. (4) Using multiple rounds of selection can be counterproductive. We conclude that each of these parameters should be taken into consideration when screening AAV libraries for enhanced properties of interest.
Assuntos
Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Biodiversidade , Linhagem Celular , Evolução Molecular Direcionada , Biblioteca Gênica , Técnicas de Transferência de Genes , Genoma Viral , Células HaCaT , Vírus Auxiliares , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reprodutibilidade dos Testes , Transdução Genética , Replicação ViralRESUMO
While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional ß cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell-derived ß cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.
Assuntos
Capsídeo/química , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Animais , Células Cultivadas , Diabetes Mellitus , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Biblioteca Gênica , Técnicas de Transferência de Genes , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rß into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Engenharia Celular/métodos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Interleucina-2/imunologia , Animais , Células HEK293 , Humanos , Melanoma Experimental , Camundongos , Receptores de Interleucina-2/genéticaRESUMO
We utilized quantitative high-resolution single particle tracking to study the internalization and endosomal sorting of lipid nanoparticles (LNPs) by HeLa cells in vitro to gain a better understanding of how cells process LNPs that are used for siRNA delivery. We compared the trafficking of three formulations that have been demonstrated to deliver siRNA into cells. They were composed of either a tritratable anionic lipid, formulation of cholesterol hemisuccinate (CHEMS), or a titratatable cationic lipid formulation of 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) or a non-titratable cationic formulation lipid formulation of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). They also contained either a substantial percentage of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol and 5 mole percent 1,2-dimyristoyl-sn-glycerol-[methoxy(polyethylene glycol)-2000 (PEG-DMG). We optically measured the endosomal pH experienced by individual LNPs, observed the internalization pathways used and tracked the particles as they co-localized with fluorescent protein tags on compartment-specific proteins, during endosomal sorting to the lysosome. The data revealed significant differences in the accumulation in subcellular compartments among the three formulations, which help to explain the observed effects LNP composition exerts on in vitro delivery efficiency.
Assuntos
Lipídeos/química , Nanopartículas/química , Transporte Biológico/fisiologia , Colesterol , Sistemas de Liberação de Medicamentos , Ácidos Graxos Monoinsaturados , Corantes Fluorescentes , Células HeLa , Humanos , Metabolismo dos Lipídeos , Luciferases , Nanopartículas/metabolismo , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Propanolaminas/química , Compostos de Amônio Quaternário , RNA Interferente Pequeno , Triglicerídeos/químicaRESUMO
Liposomes improve the pharmacokinetics and safety of rapidly cleared drugs, but have not yet improved the clinical efficacy compared to the non-encapsulated drug. This inability to improve efficacy may be partially due to the non-uniform distribution of liposomes in solid tumors. The tumor extra-cellular matrix is a barrier to distribution and includes the high molecular weight glycosaminoglycan, hyaluronan (HA). Strategies to remove HA or block its synthesis may improve drug delivery into solid tumors. Orally administered methylumbelliferone (MU) is an inhibitor of HA synthesis, but it is limited by low potency and limited solubility. In this study, we encapsulate a water-soluble phosphorylated prodrug of MU (MU-P) in a liposome (L-MU-P). We demonstrate that L-MU-P is a more potent inhibitor of HA synthesis than oral MU in the 4T1 murine mammary carcinoma model using both a quantitative ELISA and histochemistry. We show that HA depletion improves the tumor distribution of liposomes computed using Mander's colocalization analysis of liposomes with the tumor vasculature. Hyaluronan depletion also increases the fraction of the tumor area positive for liposomes. This improved distribution extends the overall survival of mice treated with Doxil®.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/análogos & derivados , Ácido Hialurônico/deficiência , Himecromona/administração & dosagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Pró-Fármacos/administração & dosagem , Administração Oral , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/metabolismo , Feminino , Himecromona/química , Injeções Intravenosas , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos Endogâmicos BALB C , Fosforilação , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Pró-Fármacos/química , Solubilidade , Fatores de Tempo , Microambiente TumoralRESUMO
Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N2-dG in DNA. B[a]P-N2-dG adducts have been shown to induce a variety of mutations, notably G-->T, G-->A, G-->C and -1 frameshifts. Four stereoisomers of B[a]P-N2-dG (designated: [+ta]-;, [+ca]-, [-ta] and [-ca]) were studied by NMR in duplex 11mers in a 5'-CGC sequence context, and each adopted a different adduct conformation (Geacintov, et al. (1997) Chem. Res. Toxicol., 10, 111). Herein these four identical B[a]P-containing 11mers are built into duplex plasmid genomes and mutagenesis studied in Escherichia coli following SOS-induction. In nucleotide excision repair (NER) proficient E.coli, no adduct-derived mutants are detected. In NER deficient E.coli, G-->T mutations dominate for all four stereoisomers [+ta]-, [+ca]-, [-ta] and [-ca]-B[a]P-N(2)-dG, and mutation frequency is similar. Thus, the mutagenic pattern for these four B[a]P-N2-dG stereoisomers is the same, in spite of the fact that they adopt dramatically different conformations in ds-oligonucleotides as determined by NMR. These findings suggest that adduct conformation must be fluid enough in the 5'-CGC sequence that the duplex DNA conformation can interconvert to mutagenic and non-mutagenic conformations during lesion-bypass. A comparison of all published studies with these four B[a]P-N2-dG stereoisomers in E.coli reveals that B[a]P-N2-dG adduct stereochemistry tends to have a lesser impact on mutagenic pattern (e.g. G-->T versus G-->A mutations) than does DNA sequence context, which is discussed.