METHOD OF DNA SYNTHESIS

The present invention relates to an in vitro cell-free process for production of deoxyribonucleotides (DNAs) comprising at least one hairpin, corresponding DNA products and uses thereof, and oligonucleotides and kits useful in the process of the invention. 1. An in vitro cell-free process for production of a deoxyribonucleic acid (DNA) molecule which comprises a desired DNA sequence , the process comprising:(a) contacting an oligonucleotide immobilised on a solid support with a series of template oligonucleotides which overlap in sequence, in the presence of at least one DNA polymerase under conditions promoting template-dependent extension of the immobilised oligonucleotide to produce a first DNA strand which comprises the desired DNA sequence and a first portion of a protelomerase target sequence proximal to the solid support;(b) introducing a DNA sequence comprising a second portion of the protelomerase target sequence into a distal end of the first DNA strand, or into a second DNA strand, such that the first and second portions of the protelomerase target sequence thereby create the protelomerase target sequence proximal to the solid support; and(c) contacting the protelomerase target sequence proximal to the solid support with a protelomerase to thereby produce and release the DNA molecule from immobilisation.2. The process according to claim 1 , wherein the immobilised oligonucleotide of (a) comprises the first portion of the protelomerase target sequence.3. The process according to claim 1 , wherein the first portion of the protelomerase target sequence is introduced into the first DNA strand by template-dependent extension.4. The process according to claim 1 , wherein the DNA molecule is a single-stranded circular DNA molecule comprising at least one hairpin that comprises the first portion or the second portion of the protelomerase target sequence.5. The process according to claim 4 , wherein step (b) comprises template-dependent extension with one or more ...

CLOSED LINEAR DNA PRODUCTION

The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferably using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art. 1. A cell-free method of producing closed linear deoxyribose nucleic acid (DNA) molecules comprising:(a) contacting a template comprising a linear, double stranded DNA molecule covalently closed at each end by a portion of a protelomerase recognition sequence and comprising at least one stem loop motif with a strand-displacing polymerase under conditions promoting amplification of said template in the presence of at least one primer which is capable of binding specifically to a primer binding site within said stem loop motif; and(b) contacting DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA.2. A method as claimed in wherein a sequence of said stem loop motif comprises a central section flanked by two sequences.3. A method as claimed in wherein the sequence of the central section of the stem loop motif comprises a primer binding site.4. A method as claimed in claim 1 , wherein a sequence forming the stem loop motif is adjacent or near to the portion of the protelomerase recognition sequence within the covalently closed end of the template.5. A method as claimed in claim 1 , wherein a sequence of said stem loop motif is capable of forming a stem loop structure under conditions promoting the formation of a secondary structure.6. A method as claimed in wherein the sequence of the stem loop ...

PREPARATION OF CLOSED, LINEAR DNA

Closed linear dna production

Synthesis of dna with improved yield

The present invention relates to an improved process for synthesis of deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large scale, with an improved yield and/or with an improved efficiency. The species of cation present in the nucleotide salt as the counter-ion is critical to the yield, efficiency and fidelity of high yielding enzymatic DNA synthesis reaction. The processes herein use alternative cations as counter-ions for the ionic nucleotides, permitting the use of higher concentrations of nucleotides in DNA synthesis, and further allowing for more favourable reaction conditions to be used.

Synthesis of dna with improved yield.

La presente invención se refiere a un proceso mejorado para la síntesis de ácido desoxirribonucleico (ADN), en particular una síntesis enzimática de ADN libre de células, preferiblemente a gran escala, con un rendimiento mejorado y/o con una eficiencia mejorada. La especie de catión presente en la sal de nucleótido como el contraión es crucial para el rendimiento, eficiencia y fidelidad de la reacción de síntesis enzimática de ADN de alto rendimiento. Los procesos descritos en este documento usan cationes alternativos como contraiones para los nucleótidos iónicos, lo que permite el uso de concentraciones más altas de nucleótidos en la síntesis de ADN, y permite además que se usen condiciones de reacción más favorables.

Vetor de dna linear fechado adequado para uso como um vetor de transferência lentiviral, e, método para melhorar o título infeccioso de partículas lentivirais quando o vetor de transferência é um vetor de dna linear fechado

vetor de dna linear fechado adequado para uso como um vetor de transferência lentiviral, e, método para melhorar o título infeccioso de partículas lentivirais quando o vetor de transferência é um vetor de dna linear fechado. a presente invenção se refere a um novo vetor de dna linear fechado, que é adequado para uso na produção de partículas lentivirais. notavelmente, a presente invenção se refere a uma nova configuração do vetor, incluindo o transgene (frequentemente denominado vetor de “carga útil”), que permite que um maior rendimento de partículas lentivirais infecciosas, notavelmente um maior rendimento de partículas lentivirais transportando um transgene, seja preparado quando comparado a vetores de dna linear fechado sem esta configuração. além disso, os inventores desenvolveram aperfeiçoamentos na produção lentiviral com dna linear fechado, através da otimização das quantidades de entrada de vetores e das razões de construção. além disso, a invenção se refere a um método de geração de partículas lentivirais infecciosas usando a construção, opcionalmente em conjunto com vetores de produção aprimorados e/ou metodologia otimizada

Nucleic acid constructs and methods for their manufacture

The present invention concerns new artificially synthesized single stranded nucleic acid molecules which may be used in many applications, and templates and methods for making the same. There are a multitude of uses for single stranded nucleic acid molecules, including but not limited to vectors for the delivery of sequences (for example a gene sequence, or a template for gene editing, gene knock-in or knock-down) or in bioengineering, for example as for constructing highly ordered materials from nanoparticle building blocks.

Self-targeting expression vector

The present invention concerns new nucleic acid molecules which may be used in many applications, and methods for making the same. These nucleic acid molecules are preferably DNA vectors, optionally DNA expression vectors. The nucleic acid molecules are able to target the vector to a specific cellular location, such as the nucleus, due to the presence of one or more particular binding motifs within the nucleic acid molecule itself. Thus, the nucleic acid molecules of the invention may also be described as targeted delivery vectors, notably self-targeted delivery vectors or smart delivery vectors.

Lentiviral vector

The present invention relates to a novel closed linear DNA vector, which is suitable for use in the production of lentiviral particles. Notably, the present invention relates to a new configuration of the vector including the transgene (often termed the "payload" vector), which enables a greater yield of infectious lentiviral particles, notably a greater yield of lentiviral particles carrying a transgene, to be prepared when compared to closed linear DNA vectors lacking this configuration. Further, the inventors have developed improvements in lentiviral production with closed linear DNA, through optimisation of vector input quantities and construct ratios. The invention furthermore relates to a method of generating infectious lentiviral particles using the construct, optionally in conjunction with improved production vectors and/or optimised methodology.

A nucleic acid delivery vector comprising a circular single stranded polynucleotide

The invention relates to a delivery vector for the delivery of a single-stranded nucleic acid. Said vector is a closed circular polynucleotide comprised of at least three sections, two of which have sufficient complementarity to form a duplex, and an intervening sequence containing the single-stranded nucleic acid to be delivered. Said duplex includes a recognition sequence for a targeted nuclease such that under appropriate conditions the single-stranded nucleic acid is released.

Vetor de entrega de ácido nucleico, método para fornecer um ácido nucleico linear de fita simples a uma célula, e, método para fornecer um modelo de doador de fita simples linear a uma célula

vetor de entrega de ácido nucleico, método para fornecer um ácido nucleico linear de fita simples a uma célula, e, método para fornecer um modelo de doador de fita simples linear a uma célula. a invenção se refere a um vetor de entrega para a entrega de um ácido nucleico de fita simples. o dito vetor é um polinucleotídeo circular fechado composto por pelo menos três seções, duas das quais têm complementaridade suficiente para formar um duplex e uma sequência intermediária contendo o ácido nucleico de fita simples a ser entregue. o dito duplex inclui uma sequência de reconhecimento para uma nuclease direcionada de modo que sob condições apropriadas o ácido nucleico de fita simples seja liberado.

Construcciones de acidos nucleicos y metodos para su fabricacion.

La presente invención se refiere a nuevas moléculas de ácido nucleico de simple cadena sintetizadas artificialmente que pueden usarse en muchas aplicaciones, y moldes y métodos para producir las mismas. Hay una multitud de usos para las moléculas de ácido nucleico de simple cadena, que incluyen, pero no se limitan a vectores para el suministro de secuencias (por ejemplo, una secuencia génica o un molde para la edición de genes, introducción o desactivación de genes) o en bioingeniería, por ejemplo, en la construcción de materiales altamente ordenados a partir de bloques de construcción de nanoparticulas.

Construtos de ácido nucleico e métodos para sua fabricação

construtos de ácido nucleico e métodos para sua fabricação. a presente invenção refere-se a novas moléculas de ácido nucleico de fita simples sintetizadas artificialmente que podem ser usadas em muitas aplicações e modelos e métodos para fazer as mesmas. há uma infinidade de usos para moléculas de ácido nucleico de fita simples, incluindo, mas sem limitação, vetores para a entrega de sequências (por exemplo, uma sequência de genes ou um modelo para edição de gene, knock-in ou knock-down de gene) ou em bioengenharia, por exemplo, para a construção de materiais altamente ordenados a partir de blocos de construção de nanopartículas.

Self-targeting expression vector

The present invention concerns new nucleic acid molecules which may be used in many applications, and methods for making the same. These nucleic acid molecules are preferably DNA vectors, optionally DNA expression vectors. The nucleic acid molecules are able to target the vector to a specific cellular location, such as the nucleus, due to the presence of one or more particular binding motifs within the nucleic acid molecule itself. Thus, the nucleic acid molecules of the invention may also be described as targeted delivery vectors, notably self-targeted delivery vectors or smart delivery vectors.

Vector de expresion autofocalizado.

La presente invención se refiere a nuevas moléculas de ácido nucleico que pueden usarse en muchas aplicaciones, y a métodos para fabricarlas. Estas moléculas de ácido nucleico son preferentemente vectores de ADN, opcionalmente vectores de expresión de ADN. Las moléculas de ácido nucleico son capaces de focalizar el vector a una ubicación celular específica, como el núcleo, debido a la presencia de uno o más motivos de unión particulares dentro de la propia molécula de ácido nucleico. Así pues, las moléculas de ácido nucleico de la invención también pueden describirse como vectores de administración focalizados, en particular vectores de administración autodirigidos o vectores de administración inteligentes.

Self-targeting expression vector

The present invention concerns new nucleic acid molecules which may be used in many applications, and methods for making the same. These nucleic acid molecules are preferably DNA vectors, optionally DNA expression vectors. The nucleic acid molecules are able to target the vector to a specific cellular location, such as the nucleus, due to the presence of one or more particular binding motifs within the nucleic acid molecule itself. Thus, the nucleic acid molecules of the invention may also be described as targeted delivery vectors, notably self-targeted delivery vectors or smart delivery vectors.

Lentiviral vector

The present invention relates to a novel closed linear DNA vector, which is suitable for use in the production of lentiviral particles. Notably, the present invention relates to a new configuration of the vector including the transgene (often termed the "payload" vector), which enables a greater yield of infectious lentiviral particles, notably a greater yield of lentiviral particles carrying a transgene, to be prepared when compared to closed linear DNA vectors lacking this configuration. Further, the inventors have developed improvements in lentiviral production with closed linear DNA, through optimisation of vector input quantities and construct ratios. The invention furthermore relates to a method of generating infectious lentiviral particles using the construct, optionally in conjunction with improved production vectors and/or optimised methodology.

Lentiviral vector

The present invention relates to a novel closed linear DNA vector, which is suitable for use in the production of lentiviral particles. Notably, the present invention relates to a new configuration of the vector including the transgene (often termed the "payload" vector), which enables a greater yield of infectious lentiviral particles, notably a greater yield of lentiviral particles carrying a transgene, to be prepared when compared to closed linear DNA vectors lacking this configuration. Further, the inventors have developed improvements in lentiviral production with closed linear DNA, through optimisation of vector input quantities and construct ratios. The invention furthermore relates to a method of generating infectious lentiviral particles using the construct, optionally in conjunction with improved production vectors and/or optimised methodology.

Lentiviral vector

The present invention relates to a novel closed linear DNA vector, which is suitable for use in the production of lentiviral particles. Notably, the present invention relates to a new configuration of the vector including the transgene (often termed the "payload" vector), which enables a greater yield of infectious lentiviral particles, notably a greater yield of lentiviral particles carrying a transgene, to be prepared when compared to closed linear DNA vectors lacking this configuration. Further, the inventors have developed improvements in lentiviral production with closed linear DNA, through optimisation of vector input quantities and construct ratios. The invention furthermore relates to a method of generating infectious lentiviral particles using the construct, optionally in conjunction with improved production vectors and/or optimised methodology.

Self-targeting expression vector

The present invention concerns new nucleic acid molecules which may be used in many applications, and methods for making the same. These nucleic acid molecules are preferably DNA vectors, optionally DNA expression vectors. The nucleic acid molecules are able to target the vector to a specific cellular location, such as the nucleus, due to the presence of one or more particular binding motifs within the nucleic acid molecule itself. Thus, the nucleic acid molecules of the invention may also be described as targeted delivery vectors, notably self-targeted delivery vectors or smart delivery vectors.

Vector lentiviral.

La presente invención se refiere a un vector de ADN lineal cerrado novedoso, el cual es adecuado para el uso en la producción de partículas lentivirales. En particular, la presente invención se refiere a una nueva configuración del vector que incluye el transgen (llamado frecuentemente el vector de "carga útil"), la cual hace posible un mayor rendimiento de partículas lentivirales infecciosas, en especial un mayor rendimiento de partículas lentivirales que llevan un transgen, que se preparan cuando se comparan con vectores de ADN lineal cerrad que carecen de esta configuración. Además, los inventores han desarrollado mejoramientos en la producción lentiviral con ADN lineal cerrado, a través de la optimización de cantidades de entrada de vectores y relaciones de constructos. La invención se refiere adicionalmente a un método para generar partículas lentivirales infecciosas por medio del uso del constructo, opcionalmente en conjunto con vectores de producción mejorados y/o metodología optimizada.