Retrovirus-based Vectors Research Articles

Efficient replication of full-length murine leukemia viruses modified at the dimer initiation site regions

Retroviruses encapsidate two copies of full-length viral RNA molecules linked together as a dimeric genome. RNA stem loop structures harboring palindromic (or “kissing”) loop sequences constitute important cis-elements for viral dimerization known as dimer initiation sites (DIS). In murine leukemia virus (MLV), a 10-mer and a 16-mer palindrome (DIS-1 and DIS-2, respectively) located in the viral leader region mediate dimerization in vitro and affect dimer stability of vector RNA in vivo. We have investigated the effect on viral replication of introducing deletions or nucleotide substitutions within these palindromes in a full-length MLV genome. Our results demonstrate that viruses modified at the dimer initiation site regions are viable and show wild-type levels of RNA encapsidation. One mutant lacking the DIS-1 palindrome was severely impaired and displayed an increased cellular ratio of spliced versus genomic RNA that most likely contributes to the inefficient replication. The implications for development of DIS-modified retrovirus-based vectors are discussed.

Retroviruses as tools to study the immune system

Retrovirus-based vectors provide an efficient means to introduce and express genes in cells of the immune system and have become a popular tool to study immune function. They are easy to manipulate and provide stable, long-term gene expression because they integrate into the genome. Current retroviral vectors do have limitations that affect their usefulness in certain applications. However, recent advances suggest a number of ways in which these vectors might be improved to extend their utility in immunological research.

Attenuation of Hypertension by Systemic Delivery of Retroviral Vector Containing Type I Angiotensin II Receptor Antisense cDNA

Despite recent strides in the traditional pharmacological therapies in the control and management of hypertension, a successful prevention and cure for this disease by conventional drug strategy remain at a standstill. We have begun to investigate the conceptual possibility of the use of gene therapy in the control of hypertension. In this article we describe an experimental protocol that provides proof of the principle that antisense (AS) inhibition of Type I angiotensin II receptor (AT1-R) could prevent development of hypertension on a long-term basis. A retrovirus-based vector has been used to deliver AT1R–AS with high efficiency that attenuates development of high blood pressure and hypertension-associated cardiac and vascular pathophysiology in the spontaneously hypertensive rat.

Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components. These reduced-homology PCLs allowed for the use of a novel high multiplicity of transduction ("high m.o. t.") method to introduce multiple copies of provector within vector-producing cell lines (VPCLs), resulting in high-titer vector without the generation of RCR. In a distinct approach to increase vector yields, we integrated manufacturing parameters into screening strategies and clone selection for large-scale vector production. Collectively, these improvements have resulted in the development of diverse VPCLs with unprocessed titers exceeding 2 x 10(7) CFU/ml. Using this technology, human Factor VIII VPCLs yielding titers as high as 2 x 10(8) CFU/ml unprocessed supernatant were generated. These cell lines produce complement-resistant vector particles (N. J. DePolo et al., J. Virol. 73: 6708-6714, 1999) and provide the basis for an ongoing Factor VIII gene therapy clinical trial.

Split-intron retroviral vectors: enhanced expression with improved safety.

The inclusion of retrovirus-derived introns within retrovirus-based expression vectors leads to a fraction of the resulting transcripts being spliced. Such splicing has been shown to markedly improve expression (W. J. Krall et al., Gene Ther. 3:37-48, 1996). One way to improve upon this still further might involve the use of more efficient introns instead of those from the provirus. Currently, however, incorporation of such introns remains self-defeating since they are removed in the nucleus of the producer cell. In the past, elaborate ways to overcome this problem have included the use of alphaviruses to make the vector transcripts within the cytoplasm, thus avoiding the nuclear splicing machinery during vector production (K. J. Li and H. Garoff, Proc. Natl. Acad. Sci. USA 95:3650-3654, 1998). We now present a novel design for the inclusion of introns within a retroviral vector. In essence, this is achieved by exploiting the retroviral replication process to copy not only the U3 promoter but also a synthetic splice donor to the 5'-long-terminal-repeat position during reverse transcription. Once copied, synthesized transcripts then contain a splice donor at their 5' end capable of interacting with a consensus splice acceptor engineered downstream of the packaging signal. Upon transduction, we demonstrate these vectors to produce enhanced expression from near fully spliced (and thus packaging signal minus) transcripts. The unique design of these high titer and high-expression retroviral vectors may be of use in a number of gene therapy applications.

Gene therapy for adenosine deaminase deficiency.

The clinical gene therapy trials for adenosine deaminase (ADA) deficiency have defined both the potential benefits and the present limitations of gene therapy with hematopoietic stem cells (HSC). Current clinical results indicate that (a) both umbilical cord blood and neonatal bone marrow HSC can be transduced with murine retroviral-based vectors, (b) the transduced HSC can engraft in nonmyeloablated patients, (c) the frequency of HSC transduction/engraftment is low (1/10,000), (d) an in vivo selective advantage can exist for transduced T lymphoid progeny, and (e) the transduced ADA gene is not expressed in nondividing T lymphocytes. Improving the clinical results of gene therapy for ADA deficiency and other genetic diseases involving HSC will require (a) developing new vectors that express the transduced gene in nondividing cells and (b) increasing the frequency of stable HSC transduction.

39] Retrovirally mediated delivery of angiotensin II type 1 receptor antisense in vitro and in vivo

In spite of excellent drugs that are available for the control of hypertension, the pharmacological approach has major disadvantages including compliance, side effects, and inability to cure the disease. In the present chapter we provide evidence that a gene therapy concept based on the inhibition of the RAS at a genetic level, with the use of an antisense to the AT1R, is an exciting and viable approach for long-term control of hypertension without the disadvantages inherent in pharmaceutical therapy. A retrovirus-based vector has been used to deliver AT1R-AS in Ang II target tissues both in vitro and in vivo. The transduction efficiency is high and leads to the attenuation of Ang II action in vitro and prevention of hypertension in the SH rat, a model for primary human hypertension. These studies have unveiled a new avenue in which a similar approach could be attempted in the reversal of hypertension in adult animals.

A High-Titer Lentiviral Production System Mediates Efficient Transduction of Differentiated Cells Including Beating Cardiac Myocytes

Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and long term transgene expression. We used a three-plasmid expression system to generate pseudotyped lentivirus-based vectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated versatile high titer lentivirus at titers of up to 2×108transducing units/ml (TU/ml), and improved transduction efficiency in various cell types from seven to over twenty fold. We demonstrate its applicability of these vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers. While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3±1.2)×105TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9±2.1)×102TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of β-galactosidase ( β-Gal) at approximately 30–40% of the maximum expression levels achieved in replicating myoblasts. In contrast, the expression ofβ-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases.

In Vivo Marking of Rhesus Monkey Lymphocytes by Adeno-Associated Viral Vectors: Direct Comparison With Retroviral Vectors

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.

Retroviral gene transfer to the liver in vivo during tri-iodothyronine induced hyperplasia.

The liver is an important target organ for gene therapy but its mitotic quiescence makes it resistant to integrative gene transfer. Retrovirus-based vectors integrate into liver cells in vivo but require the liver to be primed before transduction; experimentally a 70% hepatectomy is commonly used to stimulate regeneration, rendering the liver susceptible to transduction during the resulting wave of cell proliferation. Our aim was to develop a clinically acceptable method of inducing hepatocyte replication before in vivo retroviral gene transfer which is both simple and effective. We have used the physiological hormone tri-iodothyronine (T3) to stimulate hepatocyte replication. A single dose of T3 (400 micrograms/100 g bw) was given subcutaneously to euthyroid rats. This produced a labelling index of 31.7% in the hepatocyte population without histological or biochemical evidence of preceding liver damage. Following T3 administration the rat livers were transfected in vivo with an amphotropic retrovirus, TELCeB/AF-7 which encodes the beta-galactosidase reporter gene together with a nuclear localisation signal. Transgene expression was noted only within the liver where 1.3% of hepatocytes expressed the beta-galactosidase enzyme. This compared to 5.2% of hepatocytes transduced following a 70% hepatectomy, and 0.02% in animals receiving neither T3 nor partial hepatic resection before transduction. T3 administration is a simple way to prime the liver before in vivo retroviral vector-based gene transfer.

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.

Multigene antiviral vectors inhibit diverse human immunodeficiency virus type 1 clades.

The chronicity of infection by the human immunodeficiency virus (HIV) calls for therapeutic regimens that offer sustained antiviral effects, such as gene therapy. Recent studies have demonstrated that expression of HIV mutant transdominant proteins, RNA decoys, and ribozymes efficiently inhibited HIV replication. We have previously shown that an RNA decoy (stem-loop II of the Rev response element of HIV type 1 [HIV-1], named SL2) and a ribozyme (Rz) targeting the U5 region of the HIV-1 5' long terminal repeat (LTR), combined in a fusion molecule, was more efficient in inhibiting HIV-1 replication than the ribozyme or the decoy alone. In this study, we expressed this fusion molecule in a retrovirus-based double-copy vector to obtain higher expression of this molecule. Furthermore, we inserted a sequence internally to drive expression of another fusion molecule with a ribozyme targeting the env/rev region linked to SL2 to obtain a triple-copy vector. These multigene antiviral vectors were subsequently transduced or transfected into human CD4+ T cells (Molt-4). Results showed that the translocation of the SL2-Rz cassette from the 3' to the 5' LTR occurred in 80% of the transduced cells. The numbers of ribozyme RNA transcripts, estimated by competitive-quantitative reverse transcription (RT)-PCR, were 1.2 x 10(5), 1.2 x 10(4), and 1.5 x 10(3) copies per cell for the triple-copy, double-copy, and single-copy vectors, respectively. Cell challenge with multiple subtypes of HIV-1 (clades A to E) showed commensurate levels of virus inhibition for the three vectors. This study suggests that the combination of multiple anti-HIV genes, such as ribozymes and decoys, targeting multiple sites of HIV RNA and expressed at high levels are promising for the treatment of HIV-1 infection.

Transfection of the Escherichia coli nth Gene into Radiosensitive Chinese Hamster Cells: Effects on Sensitivity to Radiation, Hydrogen Peroxide, and Bleomycin Sulfate

The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.

Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets

Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV-associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.

Internalization of Human Immunodeficiency Virus Type I and Other Retroviruses by Megakaryocytes and Platelets

Abstract Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV- associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.

Gene expression from transcriptionally disabled retroviral vectors.

Retroviral vectors are used for the efficient transfer of foreign genes into mammalian cells. We report here the construction of murine retrovirus-based vectors carrying the full-length cDNA for human hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and from which the enhancer sequences, the "CAAT box," and the "TATA box" in the long terminal repeats (LTRs) have been deleted. After infection of HPRT-deficient rat cells by the vectors, transcriptional activity from the 5' LTR was undetectable and expression of the HPRT cDNA was dependent on an internal promoter. Removal of the LTR regulatory elements increased HPRT gene expression from an internal promoter, indicating interference between the two sets of transcriptional signals. Such disabled vectors may reduce the likelihood of undesirable genetic changes through insertional mutagenesis in cells infected with retroviral vectors.