RESUMO
Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2Obl/+, with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.
Assuntos
Surdez , Perda Auditiva , Humanos , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/genética , Lipossomos , RNA Guia de Sistemas CRISPR-Cas , Perda Auditiva/genética , Perda Auditiva/terapia , Surdez/genéticaRESUMO
The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.
Assuntos
GenômicaRESUMO
The ability to control translation of endogenous or exogenous RNAs in eukaryotic cells would facilitate a variety of biotechnological applications. Current strategies are limited by low fold changes in transgene output and the size of trigger RNAs (trRNAs). Here we introduce eukaryotic toehold switches (eToeholds) as modular riboregulators. eToeholds contain internal ribosome entry site sequences and form inhibitory loops in the absence of a specific trRNA. When the trRNA is present, eToeholds anneal to it, disrupting the inhibitory loops and allowing translation. Through optimization of RNA annealing, we achieved up to 16-fold induction of transgene expression in mammalian cells. We demonstrate that eToeholds can discriminate among viral infection status, presence or absence of gene expression and cell types based on the presence of exogenous or endogenous RNA transcripts.
Assuntos
Biossíntese de Proteínas , RNA , Animais , Mamíferos/genética , Biossíntese de Proteínas/genética , RNA Viral/genéticaRESUMO
Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals. Using Single Molecule Arrays (Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibody-mediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in a biosafety level 2 laboratory within two hours. We used this assay to assess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for a short period of time and show that neutralization and antibody levels increase over time. We also adapted the assay for SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy, COVID-19 infected, and vaccinated individuals. This assay is highly adaptable for clinical applications, such as vaccine development and epidemiological studies.
Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Testes de Neutralização/métodos , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.
Assuntos
COVID-19/etiologia , COVID-19/fisiopatologia , Haptoglobinas/fisiologia , Mucosa Intestinal/fisiopatologia , Precursores de Proteínas/fisiologia , SARS-CoV-2 , Síndrome de Resposta Inflamatória Sistêmica/etiologia , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Adolescente , Antígenos Virais/sangue , Biomarcadores/sangue , COVID-19/virologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Haptoglobinas/antagonistas & inibidores , Humanos , Lactente , Recém-Nascido , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/virologia , Masculino , Oligopeptídeos/farmacologia , Permeabilidade/efeitos dos fármacos , Estudo de Prova de Conceito , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/sangue , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Síndrome de Resposta Inflamatória Sistêmica/virologia , Adulto JovemRESUMO
Delivery into mammalian cells remains a significant challenge for many applications of proteins as research tools and therapeutics. We recently reported that the fusion of cargo proteins to a supernegatively charged (-30)GFP enhances encapsulation by cationic lipids and delivery into mammalian cells. To discover polyanionic proteins with optimal delivery properties, we evaluate negatively charged natural human proteins for their ability to deliver proteins into cultured mammalian cells and human primary fibroblasts. Here we discover that ProTα, a small, widely expressed, intrinsically disordered human protein, enables up to ~10-fold more efficient cationic lipid-mediated protein delivery compared to (-30)GFP. ProTα enables efficient delivery at low- to mid-nM concentrations of two unrelated genome editing proteins, Cre recombinase and zinc-finger nucleases, under conditions in which (-30)GFP fusion or cationic lipid alone does not result in substantial activity. ProTα may enable mammalian cell protein delivery applications when delivery potency is limiting.
Assuntos
Edição de Genes/métodos , Lipossomos/química , Proteínas/química , Edição de Genes/instrumentação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Integrases/química , Integrases/genética , Integrases/metabolismo , Lipossomos/metabolismo , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismoRESUMO
The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.
Assuntos
DNA Nucleotidiltransferases/genética , DNA/genética , Edição de Genes/métodos , Genoma Humano , Integrases/genética , Sequência de Bases , Clonagem Molecular , DNA/metabolismo , DNA Nucleotidiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Integrases/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação GenéticaRESUMO
Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.
Assuntos
Proteínas Associadas a CRISPR/administração & dosagem , Edição de Genes/métodos , Genes Dominantes/genética , Terapia Genética/métodos , Perda Auditiva/genética , Estimulação Acústica , Alelos , Animais , Animais Recém-Nascidos , Limiar Auditivo , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/uso terapêutico , Sistemas CRISPR-Cas , Sobrevivência Celular , Cóclea/citologia , Cóclea/metabolismo , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Fibroblastos , Células Ciliadas Auditivas/citologia , Perda Auditiva/fisiopatologia , Perda Auditiva/prevenção & controle , Humanos , Lipossomos , Masculino , Proteínas de Membrana/genética , Camundongos , Reflexo de SobressaltoRESUMO
Multiplex genome editing is the simultaneous introduction of multiple distinct modifications to a given genome. Though in its infancy, maturation of this field will facilitate powerful new biomedical research approaches and will enable a host of far-reaching biological engineering applications, including new therapeutic modalities and industrial applications, as well as "genome writing" and de-extinction efforts. In this Perspective, we focus on multiplex editing of large eukaryotic genomes. We describe the current state of multiplexed genome editing, the current limits of our ability to multiplex edits, and provide perspective on the many applications that fully realized multiplex editing technologies would enable in higher eukaryotic genomes. We offer a broad look at future directions, covering emergent CRISPR-based technologies, advances in intracellular delivery, and new DNA assembly approaches that may enable future genome editing on a massively multiplexed scale.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Genoma/genética , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Eucariotos/genética , Edição de Genes/métodos , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
We describe the development of 'recCas9', an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA/genética , DNA/metabolismo , Endonucleases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , DNA Nucleotidiltransferases/genética , Endonucleases/genética , Ordem dos Genes , Genoma Humano , Humanos , Modelos Biológicos , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Recombinação Genética , Deleção de SequênciaRESUMO
Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.
Assuntos
Endonucleases/genética , Genoma/efeitos dos fármacos , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Células Cultivadas , Endonucleases/efeitos dos fármacos , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Bibliotecas de Moléculas Pequenas , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
The pathogenesis and transition of normal urothelium into bladder carcinoma are multifactorial processes. Chronic inflammation causes initiation and progression of the underlying pathophysiology of invasive and metastatic cancer. A dichotomy is observed in the role of immune cells in bladder cancer. While the immune response defends the host by suppressing neoplastic growth, several immune cells, including neutrophils, macrophages and T-lymphocytes, promote tumor development and progression. The levels of human neutrophil peptide-1, -2 and -3, produced by neutrophils, increase in bladder cancer and might promote tumor angiogenesis and growth. The effect of macrophages is primarily mediated by pro-inflammatory cytokines, IL-6 and TNF-α. In addition, the underlying immunological mechanisms of two treatments, BCG and cytokine gene-modified tumor vaccines, and future directions are critically discussed.
Assuntos
Vacinas Anticâncer , Leucócitos , Mycobacterium bovis , Neoplasias da Bexiga Urinária , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Humanos , Interleucina-6/imunologia , Leucócitos/imunologia , Leucócitos/patologia , Fator de Necrose Tumoral alfa/imunologia , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/terapia , alfa-Defensinas/imunologiaRESUMO
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
Assuntos
Lipídeos/administração & dosagem , Proteínas/administração & dosagem , Cátions , Técnicas In Vitro , Transativadores/administração & dosagem , TransfecçãoRESUMO
Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites â¼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Endonucleases/genética , Edição de Genes/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases de Sítio Específico do Tipo II/química , Endonucleases/química , Genoma Humano , Humanos , Multimerização Proteica , RNA/genética , Proteínas Recombinantes de Fusão/químicaRESUMO
Supercharged proteins (SCPs) can deliver functional macromolecules into the cytoplasm of mammalian cells more potently than unstructured cationic peptides. Thus far, neither the structural features of SCPs that determine their delivery effectiveness nor their intracellular fate postendocytosis, has been studied. Using a large set of supercharged GFP (scGFP) variants, we found that the level of cellular uptake is sigmoidally related to net charge and that scGFPs enter cells through multiple pathways, including clathrin-dependent endocytosis and macropinocytosis. SCPs activate Rho and ERK1/2 and also alter the endocytosis of transferrin and EGF. Finally, we discovered that the intracellular trafficking of endosomes containing scGFPs is altered in a manner that correlates with protein delivery potency. Collectively, our findings establish basic structure-activity relationships of SCPs and implicate the modulation of endosomal trafficking as a determinant of macromolecule delivery efficiency.
Assuntos
Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Endocitose , Fluorescência , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Relação Estrutura-AtividadeRESUMO
Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Substâncias Macromoleculares/isolamento & purificação , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Eletricidade Estática , Animais , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/isolamento & purificação , Peptídeos Penetradores de Células/metabolismo , Endotoxinas/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Substâncias Macromoleculares/administração & dosagem , Substâncias Macromoleculares/metabolismo , Camundongos , Permeabilidade , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas/síntese química , Proteínas/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The use of a hyperthermal hydrogen induced cross-linking process to prepare laminates comprising polypropylene, poly(isobutylene-co-isoprene), and poly(vinyl acetate) is described. In this new, milder alternative to conventional plasma techniques, neutral molecular hydrogen projectiles were used to create carbon radicals on impacted surfaces by collision-induced dissociation of C-H bonds, and this process was used to cross-link polymers on a polypropylene surface. It was demonstrated that multiple layers of cross-linked materials could be added, creating polymer laminates with each layer introducing new functionalities and properties. In particular, the present work shows that the process is largely nondestructive toward ester functionalities. First, the esters were grafted to become nonleachable. Then, the esters were subsequently hydrolyzed to convert the surface from hydrophobic to hydrophilic. Afterward, the esters could be recovered by simple esterification demonstrating that further chemical transformations were possible.
Assuntos
Butadienos/química , Físico-Química , Ésteres/química , Hemiterpenos/química , Hidrogênio/química , Pentanos/química , Polienos/química , Polímeros/química , Polipropilenos/química , Polivinil/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Espectroscopia Fotoeletrônica , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
We discovered a class of naturally occurring human proteins with unusually high net positive charge that can potently deliver proteins in functional form into mammalian cells both in vitro and also in murine retina, pancreas, and white adipose tissues in vivo. These findings represent diverse macromolecule delivery agents for in vivo applications, and also raise the possibility that some of these human proteins may penetrate cells as part of their native biological functions.