EL CIERRE DE LOS COMEDORES COMUNITARIOS.

Once a gene has been selected to be transferred, the DNA must be recombined into a form w hich is likely to be expressed in the target cell. To this end a number of d iffe re n t techniques have been developed to transport the DNA and these tra nspo rt systems are called vectors. The entry of the DNA in the target cell is governed by the efficiency of delivery of the gene to the target cell, the physical properties of the vecto r and the time of exposure to the target. The vector carrying the DNA m ust firs t attach to and then cross the cell membrane, traverse the cytoplasm evading lysosomal digestion, and enter the nucleus where initiation of transcription of the DNA strand takes place. Current vectors used to aid DNA transport include those that make use of naturally occurring system s w hich insert foreign DNA into cells in disease states, i.e. the viral vectors, and man- made transfer system s such as the cationic liposomes (See Figure 2).

Liposom es Plasm id

O r.O

r \

Figure 2 Schematic diagram of Gene Delivery Vectors

V ecto r

Retrovirus

G ene Tran sfer

efficie n cy

0.1

D uration of

expression in vivo

S a fe ty Insertional Clinical trials

m utagenesis risk approved

1 2 m onths 9 3

Adenovirus 100 2 -4 weeks

Liposomes 1-5 2 -4 weeks 16

Plasmids 2 -4 weeks

A deno no co m p arative 2 -4 w eeks -t- -k -t- 1

associated virus data

The adenovirus, fo r example, has a specific receptor w hich enables entry into the cell, and viral coat proteins w hich disrupt the intracellular host defenses th a t norm ally cause destruction of the viral DNA and the gene of interest. Liposomes fuse w ith the cell membrane and release sufficie nt DNA into the cytoplasm , so that even w ith o u t a protective transfer mechanism ample DNA reaches the nucleus. All viral vectors used in gene therapy have been rendered replication deficient so tha t they can infect the host cell but cannot then replicate. This is accomplished by cutting specific genes out of the viruses w hich are essential fo r their replication. The replication deficient ve cto r is then grow n in cells w hich have been modified to express the deficient genes, i.e. they are com plem entary to the deleted virus. This allows large amounts of the vecto r to be produced but once it is separated from the com plem enting cells it can no longer replicate. Once DNA reaches the nucleus it is only transcribed if it has the appropriate initiation signals. In addition, the gene of interest can be combined w ith a pow erful prom oter, so tha t a therapeutic e ffe ct may be achieved w ith lesser am ounts of the vecto r and a potential reduction in to x ic ity . Tissue specific prom oters are also being investigated, e.g. an actin prom oter when targeting sm ooth muscle cells, but they often achieve sp e cificity at the expense of efficiency. The duration of expression of a gene may also determine its uses, and is often governed by the vecto r used to administer it. Genes delivered using liposomes or adenoviruses do not integrate into the host genome and therefore have a lim ited life span, w h ils t retrovirally- adm inistered genes are inserted into the genome and are, in theory, limited only by the life of the infected cell or its progeny. There is, however, no external control of the genomic site into w hich the DNA is inserted, raising the theoretical risk of insertional mutagenesis, where a native gene may be disrupted or separated from its normal control elements. The properties of some of the com m only used gene transfer vectors w hich have governed their usage are show n in Table 3. In addition to the vectors show n, viral conjugate vectors have been used, w hich aim to utilize the low to x ic ity of

liposomes w ith the efficiency of the viral vectors by linking these com ponents using polylysine 'bridges'.

1.7.2 The A denoviruses

Adenoviruses are the most w idely used viral vecto r in vascular biology. The adenovirus genome is very well characterized and is relatively easy to manipulate. This has led to the developm ent of a number of different adenoviral vectors w hich have been made replication incom petent by removal of essential genes (Graham and Prevec, 1991). Adenoviruses deleted in this w ay can accommodate the insertion of relatively large genes (8.3 kilobases), and high viral stocks can readily be grow n in the laboratory (Brett et aL 1994). They also are efficie nt at infecting many different cell types in many species. An im portant advantage over the retroviruses is their ability to infect quiescent non-dividing cells. They are highly efficient in transferring genes due to their cell surface receptor and endosomal disrupter. Their efficiency is superior to retroviruses and liposomes in

normal, uninjured and atherosclerotic blood vessels (Lemarchand e t aL

1993; Guzman e t aL 1993; Feldman e t aL 1995). Adenoviruses also have a safety advantage over retroviral vectors in tha t they do not insert their DNA into the host chromosomes, and w hile this reduces their duration of action, it also reduces the chances of insertional m utagenesis. Insertion of viral genes into the host genome can occur but is rare (Ali e t aL 1994). Use of live adenoviruses as vaccines over some years has not revealed an

increased frequency of m alignancy and is safe even in

imm unocom prom ised subjects (Rhoads e t aL 1991). The principal problem w ith adenoviruses is their ability to provoke a humoural and cellular immune response w hich may result in destruction of the infected cell. This lim its expression of the transferred gene. In addition they cause production of circulating neutralizing antibodies w hich prevent successful repeat adm inistrations (Wilson, 1996).