RESUMEN

We have studied parameters affecting in vivo expression of human growth hormone (hGH) in mice after intravenous administration of a retroviral vector encoding the protein as a model system for clotting factor VIII gene therapy. Such treatment results in a brief burst of high-level expression followed by lower level sustained expression of the hGH in the circulation. The major targets for transduction in the mouse are liver and spleen. Such direct transduction (i.e., without surgical or chemical induction of cell division) requires vector at high titer (>/=10(8) cfu/ml) and is dose dependent. Transduction efficiency decreases with increasing age of the recipient. Nevertheless, long-term expression in adults is observed after administration of vector as a split dose on 2 consecutive days. We also show that anti-vector immune responses may enhance long-term expression and that both anti-vector and anti-transgene immunity can be modulated. This work provides a framework for the rational development of means to enhance the efficiency of retroviral vectors for use in clinical gene replacement therapy.

Asunto(s)

Antígenos/genética , Factor VII/genética , Técnicas de Transferencia de Gen , Hormona de Crecimiento Humana/biosíntesis , Retroviridae/genética , Transgenes , Factores de Edad , Animales , Células Cultivadas , Ciclofosfamida/farmacología , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética/métodos , Vectores Genéticos/genética , Hepatocitos/metabolismo , Hormona de Crecimiento Humana/genética , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Factores de Tiempo , Distribución Tisular

RESUMEN

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508-519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.

Asunto(s)

ADN Viral/inmunología , Herpes Simple/prevención & control , Simplexvirus/genética , Virus Sindbis , Vacunas Virales/inmunología , Animales , ADN Viral/genética , Vectores Genéticos , Herpes Simple/virología , Inmunización , Ratones , Ratones Endogámicos BALB C , Vacunas Virales/genética

RESUMEN

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.

Asunto(s)

Presentación de Antígeno , Técnicas de Transferencia de Gen , Vectores Genéticos , Retroviridae/genética , Traslado Adoptivo , Animales , Anticuerpos/inmunología , Antígenos CD/inmunología , Recuento de Células , Células Dendríticas/inmunología , Femenino , Regulación Viral de la Expresión Génica/genética , Genes Reporteros/genética , Inmunización , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología

RESUMEN

We have developed a novel gene transfer drug, HIV-IT(V), for the treatment of HIV infection in humans. HIV-IT(V) is a retroviral vector encoding the HIV-1 IIIB env and rev genes and a neomycin resistance marker gene (neor). We have recently reported that HIV-IT(V) administered intramuscularly to male mice localizes primarily to the site of injection. In this study, we have investigated more extensively the localization and biological activity of HIV-IT(V) administered intramuscularly to female mice. Consistent with our previous findings, retroviral DNA was detected by PCR at the site of injection. Retroviral DNA was also detected in proximal lymph nodes, a tissue not examined previously. Potential for drainage of vector particles to regional lymph nodes was indicated by experiments showing that intramuscular injection of fluorescein-labeled latex beads concentrated in the regional lymph nodes in mice. The localization of retroviral DNA to the injection site and regional lymph nodes may play a role in the induction of the HIV-specific CTL responses detected in splenocyte populations isolated from mice 21 days after injection with HIV-IT(V).

Asunto(s)

ADN Viral/análisis , Vectores Genéticos/administración & dosificación , VIH-1/genética , Virus de la Leucemia Murina/genética , Ganglios Linfáticos/virología , Animales , Secuencia de Bases , ADN Recombinante/análisis , Femenino , Técnicas de Transferencia de Gen , Genes env/genética , VIH-1/inmunología , Inyecciones Intramusculares , Ganglios Linfáticos/química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Músculo Esquelético/química , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T Citotóxicos/inmunología

RESUMEN

Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.

Asunto(s)

ADN Viral , Vectores Genéticos , Virus Sindbis/genética , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Estudios de Factibilidad , Técnicas de Transferencia de Gen , Genes Reporteros , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Músculos/metabolismo , Músculos/virología , Plásmidos , ARN/biosíntesis , ARN Polimerasa II/metabolismo , ARN Viral/metabolismo , Ratas , Ratas Sprague-Dawley

Asunto(s)

ADN Recombinante/genética , Regulación Viral de la Expresión Génica , Vectores Genéticos/genética , ARN Viral/genética , Virus Sindbis/genética , Animales , Bovinos , Células Eucariotas , Genes Reporteros , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/genética , Anticuerpos contra la Hepatitis B/biosíntesis , Antígenos de la Hepatitis B/biosíntesis , Antígenos de la Hepatitis B/genética , Antígenos de la Hepatitis B/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Virus de la Leucemia Murina de Moloney/genética , ARN Polimerasa II/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Viral/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Repetitivas de Ácidos Nucleicos , Replicón , Virus Sindbis/fisiología , Replicación Viral

RESUMEN

In situ hybridization was used to document the distribution of mRNA encoding six subunit isoforms of non-N-methyl D-aspartic acid (NMDA) glutamate receptors (GluR1, GluR2, GluR3, GluR4, GluR5 and GluR6) in the inner ears of embryonic, postnatal and adult rats. GluR2 and GluR3 expression in the spiral ganglion appeared well before birth, and reached adult levels several days before the onset of function in the cochlea. In the spiral limbus, expression of GluR2 and GluR3 mRNA reached very high levels at around the time of birth, then declined after a few days. Low levels of GluR1, GluR4 and GluR6 expression were detected in various tissues of the cochlea during development. In the adult cochlea, GluR expression was limited to GluR2 and GluR3 mRNAs in the spiral ganglion neurons and GluR2 mRNA in fibrocytes of the spiral limbus, a non-neural tissue. The ontogenetic expression of additional GluR subunit genes and their appearance in different cochlear tissues could reflect different roles for these genes during development, or less precise regulation of gene expression within the GluR family. In particular, the very high levels of GluR gene expression in the spiral limbus during the perinatal period support a non-neural function, perhaps as cell surface receptors during tissue differentiation.

Asunto(s)

Cóclea/metabolismo , ARN Mensajero/análisis , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Hibridación in Situ , Ratas , Ratas Sprague-Dawley , Distribución Tisular

RESUMEN

A rat inner ear complementary DNA (cDNA) library containing 1.9 x 10(6) recombinants was constructed and evaluated. Inserts averaged 2.0 (+/- 2.1) kilobases in length. A subset of inserts was screened for site of expression. Two cDNA transcripts were isolated based on cochlear expression restricted to the spiral ganglion. One transcript showed a high degree of homology to several long interspersed DNA elements, neuron-specific nuclear transcripts thought to be involved in gene regulation. The second transcript showed no homology to known sequences and appears to encode a neuron-specific protein of about 248 amino acids. The library can be used to identify proteins important for inner ear function and disease.

Asunto(s)

Clonación Molecular , ADN Complementario/genética , Oído Interno , Biblioteca de Genes , Animales , Cóclea , Amplificación de Genes , Ratas

RESUMEN

The expression of mRNAs encoding five putative non-NMDA glutamate receptors was investigated using in situ hybridization with radiolabeled riboprobes. Hybridization was observed in spiral ganglion neurons with probes complementary to mRNA products of the glutamate receptor genes GluR2 and GluR3. No specific hybridization was observed with probes for GluR1, GluR4 or GluR5. The results support the hypothesis that glutamate is the transmitter between cochlear inner hair cells and spiral ganglion neurons, and that it acts via non-NMDA glutamate receptors.

Asunto(s)

Cóclea/química , Glutamatos/fisiología , ARN Mensajero/análisis , Receptores de Neurotransmisores/análisis , Ácido Glutámico , Células Ciliadas Auditivas Internas/química , Familia de Multigenes , Neuronas/química , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , ARN , ARN Complementario , Receptores de Glutamato , Receptores de Neurotransmisores/genética , Ganglio Espiral de la Cóclea/química

RESUMEN

Two methods for holographic cinematography are described and analyzed: the scatter-plate and the lens methods. The advantages, capabilities, and limitations of each are given.

RESUMEN

Very large bandwidths are required for the transmission of holographic data for systems such as TV. This paper presents a technique in which the large bandwidths normally required are traded off for either increased noise or decreased resolution in the image. The light radiated from the object is diffracted by an intermediate dispersion medium and collected at the hologram aperture. Correct illumination of this hologram provides an image beam that passes back through the intermediate medium and comes to focus in the space originally occupied by the object. By proper selection of the dispersion medium, the hologram aperture can be made extremely small, thus representing a large data reduction. The three dimensionality of the image and the original viewing angles are maintained. Included in the paper are experimental results that show reconstructed images after a data reduction of as much as 3600.

RESUMEN

A technique for producing many copies from one hologram is presented. This method involves using either the real or the virtual image (or both) reconstructed by a hologram as an object for a second hologram, with the undiffracted or zero-order wavefront being used as the reference beam. Such a procedure allows duplicating the first hologram without requiring close contact between the two emulsions involved. As in the recording of original holograms, a laser is used for the copying described.