Basic Principles Of Retroviral Vector Technology

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2.1. Oncoretroviral Vectors: The Basic Technology

Retroviruses are enveloped viruses that contain a diploid positive-strand RNA genome, whose life cycle is characterized by their use of reverse transcriptase to convert the RNA genome to double-stranded DNA, which is then permanently integrated into the chromosomes of the host cell. As noted above, most retroviral vectors in current use are traditionally based on Moloney murine leukemia virus (MMLV), a simple oncoretrovirus that contains 5'- and 3'-long terminal repeat (LTR) sequences flanking only three gene loci: gag, pol, and env, which encode capsid/matrix, reverse transcrip-tase/integrase, and envelope proteins, respectively (see Fig. 1). Assembly of these viral proteins to form a virion is initiated by a cis-acting sequence located next to the 5'-LTR; identification of this sequence (Y) as the dominant signal for viral packaging (see Fig. 2) enabled the development of trans-complementing systems for packaging of replication-defective viral genomes in which the natural coding sequences have been replaced by therapeutic genes of interest (see Fig. 3). Once the replication-defective vector RNA has been packaged into the nucleocapsid, the nascent virion buds from the cell surface, thereby encoating itself with the lipid bilayer membrane of the host cell, in

Table 1

Strategies for Gene Therapy of Cancer Using Retrovirus and Lentivirus Vectors

A. Ex vivo gene therapy Target cell population

Strategy

Transgenes

Hematopoietic stem cells Myeloprotection during

Hematopoietic progenitor cells high-dose chemotherapy

Hematopoietic stem cells

Cytotoxic T-lymphocytes

Cytotoxic T-lymphocytes

DCs, DC progenitor cells

(e.g., monocytes), other APCs (e.g., macrophages, Kupffer cells, microglia) Leukemia/lymphoma cells, Other explantable tumor cells

B. In vivo gene therapy Target cell population

Elimination of oncoproteins (e.g., BCR-ABL)

Abrogation of GVHD after transplantation to achieve GVL effect Tumor antigen-specific immunocytotoxicity Tumor antigen presentation, potentiation of antitumoral immune response

Tumor cell vaccine

Chemotoxin pump proteins

(e.g., MDR-1) Chemotoxin metabolizing enzymes

(e.g., DHFR, MGMT) Antisense RNA, ribozymes, siRNA directed against target mRNA Suicide genes (e.g., HSV-fi)

Tumor antigen-specific, engineered T-cell receptors Tumor-specific antigens Immunostimulatory cytokines (e.g., GM-CSF, IL-4, CD40L)

Immunostimulatory cytokines (e.

GM-CSF, IL-2, IL-12, IFNs) Costimulatory molecules (e.g., CD80, CD86, 4-1-BB)

Strategy

Transgenes

Tumor cells

Tumor cells

Tumor cells

Tumor cells

Direct cell killing by introduction of vector or vector producer cells

Over-expression of genes with tumor suppressor activity

Inhibition of oncogenes

Potentiation of antitumoral immune response

Tumor infiltrating immunocytes and APCs

DCs, DC progenitor cells (e.g., Tumor antigen

E. coli PNP) Proapoptotic genes (e.g., BAX) Toxin genes (e.g., Diphtheria toxin, HIV-vpr, hyperfusogenic GALV envelope) Tumor suppressor genes (e.g., p53, BRCA-1, p16, PTEN, MDA-7/IL-24) Cell-cycle inhibitors

(dominant-negative cyclin G1) Antisense RNA, ribozymes, siRNA directed against target mRNA Immunostimulatory cytokines (e.g.,

GM-CSF, IL-2, IL-12, IFNs) Costimulatory molecules (e.g.,

CD80, CD86, 4-1-BB) Inhibitors of immunosuppressive factors (e.g., dominant-negative TGF-P receptor) Tumor-specific antigens (e.g., monocytes), other antigen presentation, viral antitumor MART-1. NY-ESO-1)

(Continued)

Table 1 (Continued) Target cell population Strategy presenting cells (e.g., macrophages, Kupffer cells, microglia) Tumor cells Tumor endothelial cells vaccine delivered by direct in vivo injection

Inhibition of tumor neovasculature

Transgenes

Antiangiogenic factors

(e.g., endostatin, angiostatin, thrombospondin-1, soluble VEGF receptor)

Retrovirus Versus Lentivirus

Fig. 1. Comparison of oncoretrovirus vs lentivirus genomes. Both types of retrovirus are characterized by LTR sequences flanking the viral structural genes. Oncoretroviruses such as MMLV have relatively simple genome configurations, with only three structural gene loci: gag (which encodes viral capsid and matrix proteins), pol (which encodes viral protease, reverse transcriptase, and integrase proteins), and env (encoding the viral envelope protein). Lentiviruses such as HIV-1 are more complex and contain additional open reading frames encoding "accessory genes" such as tat (viral transcription factor), rev (facilitates nuclear export of viral mRNA), and various virulence factors (f: vif, r: vpr, u: vpu, n: net). Both oncoretrovirus and lentivirus genomes contain specific packaging signal sequences (y), located just downstream of the LTR and generally extending into the 5' sequence of gag, that allow encapsidation of the viral genomic RNA.

Fig. 1. Comparison of oncoretrovirus vs lentivirus genomes. Both types of retrovirus are characterized by LTR sequences flanking the viral structural genes. Oncoretroviruses such as MMLV have relatively simple genome configurations, with only three structural gene loci: gag (which encodes viral capsid and matrix proteins), pol (which encodes viral protease, reverse transcriptase, and integrase proteins), and env (encoding the viral envelope protein). Lentiviruses such as HIV-1 are more complex and contain additional open reading frames encoding "accessory genes" such as tat (viral transcription factor), rev (facilitates nuclear export of viral mRNA), and various virulence factors (f: vif, r: vpr, u: vpu, n: net). Both oncoretrovirus and lentivirus genomes contain specific packaging signal sequences (y), located just downstream of the LTR and generally extending into the 5' sequence of gag, that allow encapsidation of the viral genomic RNA.

which the viral envelope proteins are embedded (see Fig. 4). The envelope proteins mediate cellular entry by binding to receptors on the target cell surface. This binding event triggers a conformational change that activates virus-cell membrane fusion, allowing the nucleocapsid complex to be released into the cytoplasm. Reverse transcription of the viral RNA yields the double-stranded DNA proviral form, which is then permanently integrated at relatively random locations in the host cell genome, and is therefore present in all progeny cells derived from the initially infected host. With wildtype MMLV, the proviral genome then transcribes additional copies of viral genomic RNA as well as a spliced message that specifically encodes the env gene (see Fig. 2). However, with replication-defective vectors, these sequences have been replaced by therapeutic genes, which are expressed in the host cell and all progeny instead (see Fig. 4).

The generation of high titer retroviral stocks for the efficient transduction of target cells is an important technical goal for a range of gene transfer applications. This was first made feasible through the development of packaging cell lines that trans-complement vector genomes with the MMLV gag, pol, and env proteins required for virus assembly (1). Replication-defective retroviral vector genomes containing the Y encapsidation

Fig. 2. Molecular events associated with infection by a replication-competent (wild-type) retrovirus. The schematic of the infected cell on the left depicts events occurring during infection, the schematic on the right depicts events after infection has been established. 1: Virion adsorption via interaction between viral envelope protein and cell surface receptor, followed by virus-cell lipid membrane fusion. 2: Entry of viral nucleocapsid complex into cytoplasm. 3: Reverse transcription of viral genomic RNA (single line) to double-stranded DNA (double lines), U3 and U5 sequences duplicated at 5'- and 3'-ends, respectively, to convert R-U5 and U3-R into matching LTR sequences flanking viral genome. 4: Entry into cell nucleus, either by passive diffusion upon nuclear membrane breakdown during mitosis (oncoretrovirus) or uptake by active transport (lentivirus). 5: Integration of proviral DNA into host cell chromosome. 6: Transcription of viral mRNA, encoding gag, pol, and envstructural gene loci. 7: Nuclear export of viral genomic mRNA (7a), and splicing and nuclear export of viral env mRNA (7b). 8: Viral genomic sequence serves as mRNA for translation of gag and pol proteins (8a), viral env mRNA directs expression of viral envelope proteins on cell membrane. 9: Virion assembly: viral proteins encapsidate viral genomic RNA by recognition of its packaging signal (y). 10: Budding of virion from cell surface membrane, encoated by viral envelope proteins.

signal can be introduced into these packaging cells by by transient transfection with an appropriate plasmid, or in some cases by viral transduction (see Fig. 3). To obtain retro-viral stocks of the highest titers, it is necessary to establish additional virus producer cell lines that not only contain the gag-pol and env cassettes, but also have the proviral vector genome stably integrated. To identify the highest producing lines, many subclones may then need to be screened, as greatly varying titers are observed between different subclones. This screening process can take several weeks and the cell lines so established may lose their packaging ability as they are passaged. As a simpler alternative system for the production of retroviral stocks without the use of packaging lines, many groups now utilize a packaging systems for production of high titer helper-free virus stocks by transient transfection (2). Generally, human embryonic kidney-derived 293T cells are used as they are highly transfectable, and a three-plasmid transient cotransfection method is used to express: (1) a packaging plasmid expressing gag-pol;

Fig. 3. ABCs of constructing conventional replication-defective retrovirus vectors. (A) The structural genes of the virus are removed and replaced with a transgene expression cassette, which remains flanked by the viral LTR sequences. This "vector construct" retains the viral packaging signal which allows encapsidation of the vector RNA. (B) The gag-pol structural genes are generally expressed together as a "packaging construct" that can be placed under the control of a heterologous promoter (pro). (C) The viral envelope protein can be expressed by itself as a separate "envelope construct." Separating the env gene from the gag-pol genes facilitates the use of heterologous envelopes from other species to virus to encoat the vector ("pseudotyping"), and reduces the likelihood of recombination events that might lead to the reconstitution of replication-competent wildtype virus. Note that the packaging signal is deleted or mutated (Ay) in the packaging and envelope constructs so that these mRNAs cannot be encapsidated. All three constructs are expressed together within a permissive cell ("vector producer cell" or VPC) to generate the virus vector.

Fig. 3. ABCs of constructing conventional replication-defective retrovirus vectors. (A) The structural genes of the virus are removed and replaced with a transgene expression cassette, which remains flanked by the viral LTR sequences. This "vector construct" retains the viral packaging signal which allows encapsidation of the vector RNA. (B) The gag-pol structural genes are generally expressed together as a "packaging construct" that can be placed under the control of a heterologous promoter (pro). (C) The viral envelope protein can be expressed by itself as a separate "envelope construct." Separating the env gene from the gag-pol genes facilitates the use of heterologous envelopes from other species to virus to encoat the vector ("pseudotyping"), and reduces the likelihood of recombination events that might lead to the reconstitution of replication-competent wildtype virus. Note that the packaging signal is deleted or mutated (Ay) in the packaging and envelope constructs so that these mRNAs cannot be encapsidated. All three constructs are expressed together within a permissive cell ("vector producer cell" or VPC) to generate the virus vector.

(2) an envelope plasmid expressing the envelope glycoprotein env (generally the MMLV amphotropic envelope, which binds to a highly conserved inorganic phosphate transporter, PiT-2, and hence exhibits broad host species binding tropism, including human); and (3) the transfer vector plasmid expressing the replication-defective retroviral vector construct containing the gene of interest. Transient cotransfection methods can be optimized to achieve titers of up to 106 helper-free viral/mL stocks within 48 h without the need to establish and maintain packaging cell lines or stable producer lines, thereby allowing rapid production of high titer retroviral vectors for subsequent cellular trans-duction by a convenient, rapid and reproducible method, and enables rapid characterization of multiple vectors containing different genes of interest.

As vehicles for the delivery of genes into eukaryotic cells, retroviruses have several advantages (3,4): (1) gene transfer is relatively efficient, particularly in a cell culture or ex vivo setting, as most retroviral vectors are produced from packaging cells at titers on the order of 106-7 plaque-forming units (pfu)/mL; (2) stable integration into the host cell DNA is a natural part of the retroviral life cycle, and therefore the integrated provirus is passed on to all daughter cells and continues to direct the nonlytic production of its encoded products; and (3) replication-defective vectors can

Fig. 4. ABCs of constructing conventional replication-defective retrovirus vectors, part 2. Viral vector (A), packaging (B), and envelope (C) constructs are introduced into the producer cell, generally by simultaneous co-transfection for transient production of virus. Alternatively, cell lines that have been stably transfected with the packaging and/or envelope construct ("packaging cell lines"), as well as with specific vector constructs ("producer cell lines"), can be prepared for constitutive virus production. Only the RNA transcribed from the vector construct (A) contains a packaging signal (y), allowing encapsidation by the viral proteins expressed from the packaging construct (B) and envelope construct (C). The virion particle thus assembled buds from the cell surface, and because there are no viral structural genes present in the vector, only the transgene of interest is transmitted to the target cell upon infection ("vector transduction").

Fig. 4. ABCs of constructing conventional replication-defective retrovirus vectors, part 2. Viral vector (A), packaging (B), and envelope (C) constructs are introduced into the producer cell, generally by simultaneous co-transfection for transient production of virus. Alternatively, cell lines that have been stably transfected with the packaging and/or envelope construct ("packaging cell lines"), as well as with specific vector constructs ("producer cell lines"), can be prepared for constitutive virus production. Only the RNA transcribed from the vector construct (A) contains a packaging signal (y), allowing encapsidation by the viral proteins expressed from the packaging construct (B) and envelope construct (C). The virion particle thus assembled buds from the cell surface, and because there are no viral structural genes present in the vector, only the transgene of interest is transmitted to the target cell upon infection ("vector transduction").

easily be created by deletion of all essential viral genes, which renders the vectors incapable of secondary infection. An additional characteristic specific to MMLV is that it requires cell division during infection so that the nucleocapsid complex can gain access to the host cell genome, and hence cannot infect nondividing cells. As many cell types are considered to be largely quiescent in vivo, the traditional application, which has been adopted for MMLV-based retroviral vectors has been to transduce cell lines in culture; when animal studies have been performed using retroviral gene delivery, this has usually been accomplished by viral infection of primary cells in culture by the ex vivo method, followed by reimplantation of the transduced cells. This approach requires surgical acquisition, isolation, and culture of autologous cells. This is labor intensive and invasive and limits the scope of ex vivo retroviral gene transfer to those cell types that can be readily accessed, manipulated in culture, and reimplanted (e.g., hematopoietic cells, skin fibroblasts, and hepatocytes). On the other hand, this absolute selectivity for actively dividing cells results in preferential infection of malignant cells, which can be advantageous for cancer-related research and therapeutics.

2.2. Lentiviral Vectors: The Next Generation

The lentiviridae are complex retroviruses that contain additional regulatory and patho-genicity-enhancing "accessory" genes in addition to the gag, poland envstructural proteins expressed by oncoretroviruses. For human immunodeficiency virus (HIV), for example, the additional regulatory genes are tat and rev, and the pathogenicity-enhanc-ing accessory genes are vif, vpr, vpu and nef (see Fig. 1). Furthermore, although the overall life cycle of lentiviruses is similar to that of oncoretroviruses, there are several major differences. As noted above, vectors based on oncoretroviruses such as MMLV can only transduce cells that divide shortly after infection, because the MMLV preinte-gration complex cannot achieve chromosomal integration in the absence of nuclear envelope breakdown during mitosis.

In contrast, lentiviruses can infect nonproliferating cells, owing to the karyophilic properties of the lentiviral preintegration complex, which allows recognition by the cell nuclear import machinery. Correspondingly, lentiviral vectors can transduce cell lines that are growth arrested in culture, as well as terminally differentiated primary cells including hematopoietic stem cells, neurons, hepatocytes, cardiomyocytes, endothe-lium, alveolar pneumocytes, keratinocytes and dendritic cells (5-13). Hence, there has been a keen interest in the development of vector systems based on a wide variety of lentiviruses, including HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) recently. This chapter focuses primarily on HIV-based lentiviral vectors as this technology has progressed most rapidly and is already in clinical trials.

Furthermore, the possible toxicity of HIV accessory genes retained in lentiviral vector constructs, as well as the possibility of recombination leading to generation of wildtype virus, has also been raised as a safety concern. Considerable effort has been invested in the generation of more efficient and safer vectors (14,15). The lentiviral packaging system was originally developed by Naldini et al. following a tripartite transient transfection procedure (5) and later evolved into the "second generation" lentiviral vectors, where the four accessory genes of HIV (vif, vpr, vpu, and nef) were deleted from the viral packaging system without affecting viral titers or transduction efficiency (15). The only remaining auxiliary gene in this system was, therefore, rev, which, along with the Rev response element (RRE) as its cognate binding sequence, is required for efficient export of the vector and packaging construct RNAs from the nucleus during virus production. Thus both toxicity as well as the likelihood of recombination are reduced in these second- and third-generation lentiviral vector systems (see Fig. 5). More recently, further optimization of packaging systems for HIV and other lentiviruses has aimed at minimizing the risk of homologous recombination with HIV by splitting the gag-polgenes (16) and by cross-packaging configurations (i.e. using the packaging system of HIV to encapsidate transfer vectors from other lentiviral origins [SIV, HIV-2, FIV]) 117,18).

In most cases, the vectors are pseudotyped (i.e., encoated with a heterologous envelope protein) with vesicular stomatitis virus glycoprotein (VSV-G), which is a rhabdovirus envelope protein that is reported to bind to cell-surface phospholipids thereby achieving a wide host range. However, the VSV-G protein is highly fusogenic, and even with the use of inducible promoters, it has proven difficult to generate high titer stable packaging cell lines expressing VSV-G as a result of its cytotoxicity. Hence, transient transfection is the most commonly employed method for lentiviral vector production, and the use of transiently produced vectors has been approved in the first clinical trial of lentiviral gene

A Vector construct (SIN vector) pRRLsinCMViresGFP:

transgene

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