RESUMEN
Compounders worldwide are responsible for ensuring that the sterile preparations they dispense are pure, potent, and safe. To achieve that result, proper cleaning and disinfection of International Organization for Standardization controlled environments must occur. Because those tasks must be performed according to established standards, the compounding pharmacist must research regulatory requirements and appropriate products for use. In this report, we focus on U.S. regulations, guiding entities, and effective products that enable compliance with the increasingly stringent procedures required for pharmaceutical compounding. We also review cleaning and disinfecting processes, discuss the importance of correctly choosing and using disinfectants and/ or sporicidal disinfectants with surface claims in the cleanroom, and provide answers to questions frequently asked by staff who use those agents. In addition, we profile specific disinfectants that are compliant with United States Pharmacopeia Chapter <797> and current good manufacturing practice standards. Biological safety cabinets and compounding aseptic containment isolators must undergo an additional process that deactivates hazardous drug residues and removes them from the interior surfaces of those devices before they are cleaned and disinfected, but that discussion is beyond the scope of this article.
Asunto(s)
Desinfectantes , Farmacia , Humanos , Composición de Medicamentos , Desinfección , Ambiente ControladoRESUMEN
Real-world evidence suggests a trend toward inferior survival of patients receiving CD19 chimeric antigen receptor (CAR) T-cell therapy in Europe (EU) and with tisagenlecleucel. The underlying logistic, patient- and disease-related reasons for these discrepancies remain poorly understood. In this multicenter retrospective observational study, we studied the patient-individual journey from CAR-T indication to infusion, baseline features, and survival outcomes in 374 patients treated with tisagenlecleucel (tisa-cel) or axicabtagene-ciloleucel (axi-cel) in EU and the United States (US). Compared with US patients, EU patients had prolonged indication-to-infusion intervals (66 versus 50 d; P < 0.001) and more commonly received intermediary therapies (holding and/or bridging therapy, 94% in EU versus 74% in US; P < 0.001). Baseline lactate dehydrogenase (LDH) (median 321 versus 271 U/L; P = 0.02) and ferritin levels (675 versus 425 ng/mL; P = 0.004) were significantly elevated in the EU cohort. Overall, we observed inferior survival in EU patients (median progression-free survival [PFS] 3.1 versus 9.2 months in US; P < 0.001) and with tisa-cel (3.2 versus 9.2 months with axi-cel; P < 0.001). On multivariate Lasso modeling, nonresponse to bridging, elevated ferritin, and increased C-reactive protein represented independent risks for treatment failure. Weighing these variables into a patient-individual risk balancer (high risk [HR] balancer), we found higher levels in EU versus US and tisa-cel versus axi-cel cohorts. Notably, superior PFS with axi-cel was exclusively evident in patients at low risk for progression (according to the HR balancer), but not in high-risk patients. These data demonstrate that inferior survival outcomes in EU patients are associated with longer time-to-infusion intervals, higher tumor burden/LDH levels, increased systemic inflammatory markers, and CAR-T product use.
RESUMEN
Hypokalemia and hyperosmolar hyperglycemic syndrome (HHS) are two reversible but potentially fatal disorders that are important to identify and treat urgently. A 43-year-old patient presented to the ED with altered mental status and slurred speech, difficulty communicating, left-sided facial droop, and stool incontinence according to emergency medical services. This was preceded by 1.5 weeks of nausea, vomiting, polydipsia, and weight loss. On presentation, the patient was found tachycardic and tachypneic, with uncertain neurological deficits on physical exam, hyperglycemia, and electrocardiogram (EKG) abnormalities. Lab data were consistent with hyperosmolar hyperglycemic nonketotic coma. This case provides two important clinical scenarios in which cardiac EKG abnormalities and focal neurological deficits are the product of hyperosmolality and electrolyte abnormalities. Hypokalemia with EKG abnormalities consistent with a potential ischemic disease can progress into wide complex tachycardia and ventricular fibrillation. Hyperosmolar hyperglycemia may manifest with focal neurological deficits and without the classical presentation of a coma. Careful consideration of EKG and lab values in the context of clinical presentation may provide clues to resolvable etiologies. We report a case of a patient who presented to the ED with hypokalemia and HHS, both reversible but potentially fatal disorders that are important to identify and urgently treat.
RESUMEN
Patients with renal impairment (RI) are typically excluded from trials evaluating chimeric antigen receptor (CAR) T cell therapies. We evaluated the outcomes of patients with RI receiving standard of care (SOC) CAR T cell therapy for relapsed/refractory (R/R) diffuse large B cell lymphoma (DLBCL). In this retrospective, single-center cohort study of patients with R/R DLBCL treated with SOC axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) after 2 or more prior lines of therapy, renal and survival outcomes were compared based on RI and fludarabine dose reduction (DR) status. RI was defined by an estimated glomerular filtration rate <60 mL/min/1.73 m2 as determined by the Modification of Diet in Renal Disease equation using day -5 creatinine (Cr) values. Acute kidney injury (AKI) was identified and graded using standard Kidney Disease: Improving Global Outcomes criteria. Renal recovery was considered to occur if Cr was within .2 mg/mL of baseline by day +30. Fludarabine was considered DR if given at <90% of the recommended Food and Drug Administration label dose. Among 166 patients treated with CAR T cell therapy were 17 patients (10.2%) with baseline RI and 149 (89.8%) without RI. After CAR T cell infusion, the incidence of any grade AKI was not significantly different between patients with baseline RI and those without RI (42% versus 21%; P = .08). Similarly, severe grade 2/3 AKI was seen in 1 of 17 patients (5.8%) with baseline RI and in 11 of 149 patients (7.3%) without RI (P = 1). Decreased renal perfusion (28 of 39; 72%) was the most common cause of AKI, with cytokine release syndrome (CRS) contributing to 17 of 39 AKIs (44%). Progression-free survival (PFS) and overall survival (OS) did not differ between patients with RI and those without RI or between those who received standard-dose fludarabine and those who received reduced-dose fludarabine. In contrast, patients with AKI had worse clinical outcomes than those without AKI (multivariable PFS: hazard ratio [HR], 2.1; 95% confidence interval [CI], 1.2 to 3.7; OS: HR, 3.9; 95% CI, 2.1 to 7.4). Notably, peak inflammatory cytokine levels were higher in patients who experienced AKI. Finally, we describe 2 patients with end-stage renal disease (ESRD) on dialysis who received lymphodepletion and CAR T cell therapy. Baseline renal function did not affect renal or efficacy outcomes after CAR T cell therapy in DLBCL. On the other hand, patients with AKI went on to experience worse clinical outcomes. AKI was commonly related to CRS and high peak inflammatory cytokine levels. CAR T cell therapy is feasible in patients with ESRD and requires careful planning of lymphodepletion.
Asunto(s)
Lesión Renal Aguda , Fallo Renal Crónico , Linfoma de Células B Grandes Difuso , Receptores Quiméricos de Antígenos , Estados Unidos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Receptores Quiméricos de Antígenos/uso terapéutico , Estudios Retrospectivos , Estudios de Cohortes , Diálisis Renal , Antígenos CD19/efectos adversos , Linfoma de Células B Grandes Difuso/terapia , Síndrome de Liberación de Citoquinas/etiología , Lesión Renal Aguda/terapia , Fallo Renal Crónico/inducido químicamente , Riñón/fisiología , Citocinas/uso terapéutico , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
This study details a reliable and efficient method for CRISPR-Cas9 genome engineering in the high amino acid-producing strain of Corynebacterium glutamicum, NRRL-B11474. Our investigation demonstrates that a plasmid-encoded single-guide RNA paired with different edit-encoding fragments is sufficient to generate edits without the addition of an exogenous recombinase. This approach leverages a genome-integrated copy of the cas9 gene for reduced toxicity, in combination with a single plasmid carrying the targeting guide RNA and matching edit fragment. Our study systematically investigated the impact of homology arm length on editing efficiency and demonstrates genome editing with homology arm lengths as small as 25 bp for single-nucleotide polymorphisms and 75 bp for 100 bp sequence swaps. These homology arm lengths are smaller than previously reported for other strains of C. glutamicum. Our study finds that C. glutamicum NRRL-B11474 is not amenable to efficient transformation with plasmids containing the BL1, NG2, or CC1 origins of replication. This finding differs from all previously reported approaches to plasmid-based CRISPR-Cas9 or Cpf1 editing in other strains of C. glutamicum. Two alternative origins of replication (CG1 and CASE1) can be used to successfully introduce genome edits; furthermore, our data demonstrate improved editing efficiency when guide RNAs and edit fragments are encoded on plasmids carrying the CASE1 origin of replication (compared to plasmids carrying CG1). In addition, this study demonstrates that efficient editing can be done using an integrated Cas9 without the need for a recombinase. We demonstrate that the specifics of CRISPR-Cas9 editing configurations may need to be tailored to enable different edit types in a particular strain background. Refining configuration parameters such as edit type, homology arm length, and plasmid origin of replication enables robust, flexible, and efficient CRISPR-Cas9 editing in differing genetic strain contexts.
Asunto(s)
Sistemas CRISPR-Cas , Corynebacterium glutamicum/genética , Edición Génica , Eliminación de Gen , Plásmidos/genética , Polimorfismo de Nucleótido Simple , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/aislamiento & purificaciónRESUMEN
Compounders worldwide are responsible for ensuring that the sterile preparations they dispense are pure, potent, and safe. To achieve that result, proper cleaning and disinfection of International Organization for Standardization controlled environments must occur. Because those tasks must be performed according to established standards, the compounding pharmacist must research regulatory requirements and appropriate products for use. In this report, we focus on U.S. regulations, guiding entities, and effective products that enable compliance with the increasingly stringent procedures required for pharmaceutical compounding. We also review cleaning and disinfecting processes, discuss the importance of correctly choosing and using disinfectants and/ or sporicidal disinfectants with surface claims in the cleanroom, and provide answers to questions frequently asked by staff who use those agents. In addition, we profile specific disinfectants that are compliant with UnitedStates Pharmacopeia Chapter <797> and current good manufacturing practice standards. Biological safety cabinets and compounding aseptic containment isolators must undergo an additional process that deactivates hazardous drug residues and removes them from the interior surfaces of those devices before they are cleaned and disinfected, but that discussion is beyond the scope of this article.
Asunto(s)
Desinfectantes/farmacología , Composición de Medicamentos , Desinfección , HumanosRESUMEN
Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.
Asunto(s)
ADN Ligasas/metabolismo , ADN/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Clonación Molecular , ADN/química , Eliminación de Gen , Recombinación Homóloga , Mutagénesis Insercional , Polimorfismo de Nucleótido Simple , Saccharomyces cerevisiae/metabolismoRESUMEN
DNA 'assembly' from 'building blocks' remains a cornerstone in synthetic biology, whether it be for gene synthesis (â¼ 1 kb), pathway engineering (â¼ 10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.
Asunto(s)
Enzimas de Restricción del ADN , ADN/química , Biología Sintética/métodos , Algoritmos , Simulación por Computador , Electroforesis Capilar , Análisis de Secuencia de ADN , Biología Sintética/economíaRESUMEN
Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.
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Anticuerpos Antiidiotipos/inmunología , Linfocitos B/inmunología , Vacunas contra el Cáncer/inmunología , Inmunoterapia/métodos , Linfoma/inmunología , Linfoma/prevención & control , Transducción de Señal/inmunología , Animales , Antígenos CD19/inmunología , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Citometría de Flujo , Ratones , Plásmidos/genéticaRESUMEN
Escherichia coli cell-free protein synthesis (CFPS) uses E. coli extracts to make active proteins in vitro. The basic CFPS reaction mixture is comprised of four main reagent components: (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. In this work, we have simplified and shortened the protocols for preparing the CFPS chemical mixture, cell extract, and T7 RNAP. First, we streamlined the workflow for preparing the CFPS chemical solutions by combining all the chemicals into a single reagent mixture, which we call Premix. We showed that productive cell extracts could be made from cells grown in simple shake flasks, and we also truncated the preparation protocol. Finally, we discovered that T7 RNAP purification was not necessary for CFPS. Crude lysate from cells over-expressing T7 RNAP could be used without deleteriously affecting protein production. Using chloramphenicol acetyltransferase (CAT) as a model protein, we showed that these streamlined protocols still support high-yielding CFPS. These simplified procedures save time and offer greater accessibility to our laboratory's CFPS technology.
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Fraccionamiento Celular/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/genética , Transcripción GenéticaRESUMEN
We present a cell-free protein synthesis (CFPS) platform and a one-step, direct conjugation scheme for producing virus-like particle (VLP) assemblies that display multiple ligands including proteins, nucleic acids, and other molecules. Using a global methionine replacement approach, we produced bacteriophage MS2 and bacteriophage Qß VLPs with surface-exposed methionine analogues (azidohomoalanine and homopropargylglycine) containing azide and alkyne side chains. CFPS enabled the production of VLPs with yields of ~ 300 µg/mL and with 85% incorporation of methionine analogues without requiring a methionine auxotrophic production host. We then directly conjugated azide- and alkyne-containing proteins (including an antibody fragment and the granulocyte-macrophage colony stimulating factor, or GM-CSF), nucleic acids and poly(ethylene glycol) chains to the VLP surface using Cu(I) catalyzed click chemistry. The GM-CSF protein, after conjugation to VLPs, was shown to partially retain its ability to stimulate the proliferation of cells. Conjugation of GM-CSF to VLPs resulted in a 3-5-fold reduction in its bioactivity. The direct attachment scheme facilitated conjugation of three different ligands to the VLPs in a single step, and enabled control of the relative ratios and surface abundance of the attached species. This platform can be used for the production of novel VLP bioconjugates for use as drug delivery vehicles, diagnostics, and vaccines.
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Alanina/análogos & derivados , Alquinos/química , Química Clic , Glicina/análogos & derivados , Vacunas de Partículas Similares a Virus/química , Alanina/química , Animales , Antígenos/inmunología , Línea Celular , Sistema Libre de Células/metabolismo , Islas de CpG , ADN/química , ADN/genética , Glicina/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Idiotipos de Inmunoglobulinas/inmunología , Idiotipos de Inmunoglobulinas/metabolismo , Linfoma de Células B/inmunología , Ratones , Polietilenglicoles/química , Especificidad por Sustrato , Propiedades de Superficie , Vacunas de Partículas Similares a Virus/biosíntesis , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/metabolismoRESUMEN
The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment - TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.
Asunto(s)
Vacunas contra el Cáncer/genética , Escherichia coli/genética , Idiotipos de Inmunoglobulinas/genética , Linfoma de Células B/prevención & control , Fragmentos de Péptidos/genética , Toxina Tetánica/genética , Vacunas de ADN/genética , Animales , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/aislamiento & purificación , Vacunas contra el Cáncer/uso terapéutico , Femenino , Humanos , Inmunización , Idiotipos de Inmunoglobulinas/inmunología , Idiotipos de Inmunoglobulinas/aislamiento & purificación , Idiotipos de Inmunoglobulinas/uso terapéutico , Linfoma de Células B/inmunología , Ratones , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/uso terapéutico , Pliegue de Proteína , Toxina Tetánica/inmunología , Toxina Tetánica/aislamiento & purificación , Toxina Tetánica/uso terapéutico , Vacunas de ADN/inmunología , Vacunas de ADN/aislamiento & purificación , Vacunas de ADN/uso terapéuticoRESUMEN
Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.
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Copépodos/enzimología , Luciferasas/química , Luminiscencia , Proteínas Mutantes/química , Secuencia de Aminoácidos , Animales , Luciferasas/genética , Luciferasas/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , MutaciónRESUMEN
Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.
Asunto(s)
Anticuerpos Antineoplásicos/biosíntesis , Copépodos/enzimología , Región Variable de Inmunoglobulina/biosíntesis , Luciferasas/biosíntesis , Linfoma de Células B/diagnóstico , Ingeniería de Proteínas , Secuencia de Aminoácidos , Animales , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Idiotipos de Inmunoglobulinas/biosíntesis , Idiotipos de Inmunoglobulinas/genética , Idiotipos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Luciferasas/genética , Luciferasas/inmunología , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target.
Asunto(s)
Reprogramación Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Animales , Sistema Libre de Células , Células Cultivadas , ADN/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/química , Fibroblastos/metabolismo , Humanos , Factor 4 Similar a Kruppel , Ratones , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/genéticaRESUMEN
A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as approximately 500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.
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Clonación Molecular/métodos , ADN/síntesis química , Oligonucleótidos/química , Reacción en Cadena de la PolimerasaRESUMEN
Type I polyketide synthase (PKS) genes consist of modules approximately 3-6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.
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Biotecnología/métodos , Ingeniería Genética/métodos , Sintasas Poliquetidas/química , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Técnicas Químicas Combinatorias , Escherichia coli/metabolismo , Lactonas/química , Macrólidos/química , Modelos Químicos , Datos de Secuencia Molecular , Plásmidos/metabolismo , Sintasas Poliquetidas/biosíntesis , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Geldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the "inert" groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.
Asunto(s)
Antineoplásicos , Ingeniería Genética , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Sintasas Poliquetidas/genética , Quinonas , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Secuencia de Bases , Benzoquinonas , División Celular/efectos de los fármacos , Línea Celular Tumoral , Diseño de Fármacos , Regulación Bacteriana de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas , Conformación Molecular , Datos de Secuencia Molecular , Sintasas Poliquetidas/metabolismo , Quinonas/aislamiento & purificación , Quinonas/metabolismo , Quinonas/farmacología , Streptomyces/enzimología , Streptomyces/genética , Streptomyces/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440-15445]. The major current impediment to the success of this tactic is the difficulty of building the approximately 5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp "synthons" with low error frequency by automated PCR-based gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-kb segments by using only three endonucleases and "ligation by selection." These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.