Vector Transgene Capacity

The treatment of monogenic diseases requires only limited vector capacity, but complex applications may require the delivery of large or multiple independent genetic sequences. A comparison of the genome structure and capacity of several current vector systems is shown in Fig. 4. The overall size of the HSV-1 genome (152 kb) represents an attractive feature for employing the vector for the transfer of large amounts of exogenous genetic sequences. Approximately one-half of the HSV-1 coding sequences are nonessential for virus replication in cell culture and therefore may be deleted to increase transgene capacity without blocking viral replication (Fig.

Life Cycle Merino Sheep

Figure 2 HSV-1 life cycle in the host. (A) Lytic infection. Primary lytic infection of epithelial or mucosal cells results from the attachment and penetration of HSV particles to host cells, a complex process involving many HSV surface glycoproteins. Following transport of the capsid to the nuclear membrane and injection of linear dsDNA into the nucleus, the genome circularizes and begins to express the lytic HSV gene functions in a highly regulated sequential cascade, yielding the expression of proteins involved in viral DNA synthesis and virion structural components. Following assembly of newly synthesized particles within the nucleus, virion maturation results in the egress of these virions from the infected cell. (B) Schematic diagram of the sequential cascade of lytic gene expression. The 5 IE or a genes are expressed immediately upon infection through transactivation by the VP16 tegument protein. The ICP4, ICP27, and ICP0 IE gene functions are responsible for the activation of the early or p genes that are primarily involved in viral DNA synthesis. In addition, ICP4 acts to shut off expression of the IE genes. Following viral DNA replication, ICP4, ICP22, and ICP27 participate in the activation of true viral late or y genes, which mainly encode virion structural components. (C) Latent infection. When virion particles encounter and bind to axonal termini that innervate the site of primary infection, viral capsids are transported in a retrograde manner to the nerve cell body. At this point the circular viral genome can persist as an episomal molecule in a latent state within the neuron, wherein viral lytic gene expression is silenced and a series of latency-associated transcripts (LATs) are produced. (D) Gene expression during latency. The major 2.0-Kb latency-associated transcript (LAT) arises from the large 8.3-Kb polyA+ through a splicing event that yields an unstable 6.3-Kb LAT and a circular LAT lariat of 2.0 Kb. The location of the latency active promoter (LAP) regions LAP1 and LAP2 relative to the LATs is depicted.

1B). The latency region of the virus genome represents approximately 8 kb of sequence that can be removed and the joint region of the virus is composed of 15 kb of redundant sequence that can be eliminated without compromising virus replication (52). In one set of experiments we removed an 11kb section of the US region of the genome (Laquerre and Glorioso, unpublished data, 1998) containing gD, the only essential gene in this region, which can be propagated on a cell line that expresses gD in trans (4). Approximately 44 kb of HSV sequence can potentially be removed and vectors propagated in cells engineered to complement just 3 viral functions (ICP4, ICP27, and gD). Transgene expression cassettes can also be inserted into deleted essential gene loci to avoid transfer of foreign sequences to wild-type virus by recombination that could potentially occur between the vector and wild-type genomes in vivo. We have observed that some nonessential genes (e.g., IE genes ICP0 and ICP22) are toxic to some cell types, yet the products of these genes are required for high-titer vector production. The toxicity of these products makes it difficult to produce a complementing cell line carrying these genes. However, it is possible to engineer the promoters for these genes in a manner to make their function dependent on viral IE genes present only in complementing cells (44). By the judicious selection of viral gene deletions and promoter alterations, high-titer vectors can be produced with minimal complementation.

We have developed a panel of novel HSV-1 vectors with a background suitable for expression of multiple transgenes

Figure 3 Reduced toxicity following infection of primary dorsal root ganglia neurons. (A) Schematic representation of the firstgeneration SOZ.1 (ICP4", UL41":ICP0p-lacZ: UL24~:ICP4p-tk) and the third-generation TOZ.1 (ICP4", ICP22", ICP27", UL41_ :ICP0p-lacZ, UL24-:ICP4p-tk) replication-defective vectors displaying the lacZ transgene inserted into the UL41 locus under the control of the ICP0 promoter using the SV40 polyade-nylation signal. (B) The number of primary dorsal root ganglion (DRG) neurons undergoing apoptosis was determined at various times following infection with either SOZ.1 or TOZ.1. Even at an MOI of 30, TOZ.1 was less toxic than the first-generation vector (SOZ.1) at the lower MOI (3.0).

Figure 3 Reduced toxicity following infection of primary dorsal root ganglia neurons. (A) Schematic representation of the firstgeneration SOZ.1 (ICP4", UL41":ICP0p-lacZ: UL24~:ICP4p-tk) and the third-generation TOZ.1 (ICP4", ICP22", ICP27", UL41_ :ICP0p-lacZ, UL24-:ICP4p-tk) replication-defective vectors displaying the lacZ transgene inserted into the UL41 locus under the control of the ICP0 promoter using the SV40 polyade-nylation signal. (B) The number of primary dorsal root ganglion (DRG) neurons undergoing apoptosis was determined at various times following infection with either SOZ.1 or TOZ.1. Even at an MOI of 30, TOZ.1 was less toxic than the first-generation vector (SOZ.1) at the lower MOI (3.0).

using a rapid gene insertion procedure (53). To take advantage of the reduced cytotoxicity resulting from the deletion of ICP4, ICP22, and ICP27 genes (41,43), we designed a single vector in which 9 viral genes were deleted, removing a total of 11.6 kb of viral DNA that was replaced with multiple transgenes under control of different promoters. These HSV multigene vectors were constructed with either 4 or 5 independent transgenes at distinct loci (47) with all the transgenes simultaneously expressed for up to 7 days. These multigene vectors demonstrate the potential for using HSV-1 vectors for the expression of complex sets of transgenes that have coordinated or complementary functions.

Recent work inserting bacterial artificial chromosome (BAC) elements into large viruses including HSV promises to increase the speed of recombinant construction as well as allow the creation of extensively modified vectors without the necessity of creating complementing cell lines. We have introduced the BAC elements into multiply defective HSV vectors and propagated them in Escherichia coli (Wolfe and Glorioso, unpublished data, 2003). Transfection of HSV:BAC DNA purified from bacteria into complementing cells is much more efficient than using viral DNA purified from mammalian cells. Infectious virus produced can be recovered back into E. coli for further modification. Several methods have been used to modify the viral genome in bacteria with high frequencies of recombinant production in a greatly shortened time frame. We are further modifying vector genomes and engineering additional methods for recombinant engineering in bacteria.

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