Plasmid DNA Properties

The plasmid DNA used in non-viral gene delivery applications is required to possess certain qualities in terms of size, gene sequences and conformation. Plasmids, the workhorse of the biotechnology revolution, are naturally occurring entities of bacterial origin. These DNA units are found as extra chromosomal DNA within bacterial species such as E.coli (Figure 2.2) (Caldwell, 1997; Feigner, 1997; M iddaugh et a l,

1997). Plasmids are usually isolated as covalently closed circular DNA in a negative supercoiled form (i.e. the DNA is interwound, containing fewer helical turns, compared to relaxed forms, resulting in torsional tension in the plasmid loop). The conformation of the plasmid is important and for gene delivery purposes it is preferable to keep the plasmid DNA in its native conformation of negative super coiled (SC) conformation. This conformation is known to produce the highest transfection efficiency as it dissociates from and complexes with the greatest ease aiding uptake of the DNA to the cell nucleus (Adami et a l, 1998; Even-Chen and Barenholz, 1999). Conversion of the SC DNA to a circular conformation has only a minor effect but conversion to a linear state reduces the efficiency of gene expression by 90% (Figure 2.2) (Adami et a l, 1998). The natural sequence of these plasmids does not however contain all the genes required for effective gene delivery and further manipulation is required to produce a sequence suitable for effective gene delivery.

There is a limit to the size of the construct that can be packaged into the delivery system and easily inserted into the target cells. DNA macromolecules are relatively large, for example, DNA of seven kilo bases (kb) has a theoretical hydrodynamic diameter of SOOnm and the upper limit for packaging is at present 30kb (Tsai et a l,

1998). The sequences contained in the plasmid are therefore important and only essential genetic information can be included. Essential information includes the therapeutic gene, which must be identified and cloned into the appropriate expression vector, the signal sequences for gene expression such as promoters and enhancers and in some cases genes that aid integration into the genome of the diseased cell (Mhashilkar et a l, 2001). Ideally the vector should also contain an on/off switch to provide a level of control, however, currently available switches need further development (Prud’homme et a l, 2000). To limit the possibility for chromosomal

Chapter!. Literature Siir\>ey

in teg ratio n d u rin g the deliv ery o f genes to a p atien t the h o m o lo g y o f the pD N A sequ en ces to know n sequences in the hum an g e n o m e sh o u ld be ex a m in e d and d escrib ed and stro n g h o m ology avoided if p o ssib le (Z oon, 1996). T o ta ilo r p lasm id s to contain the essen tial gene sequence for p ro d u c tio n o f the d e sire d p rotein a re co m b in an t D N A te c h n o lo g y pro cess is em p lo y ed (C ald w ell, 1997). T h e ta ilo rin g p ro c ess begins w ith clea v in g the p lasm id and therapeutic gen es at precise lo c a tio n s, using restrictio n enzym es, in o rd e r to produce co m p atib le D N A en d s and the th era p eu tic genes, along w ith the a p p ro p riate p ro m o ter and e n h a n c e r seq u e n ces, can th en be lig ated into the

p lasm id via th e ir co m p atib le end (S tribley et al., 2002). T h e re su lt o f this m anip u latio n

is the p ro d u c tio n o f a sm all, well d esigned, c u sto m -b u ilt ex p ressio n vector, aiding therapeutic d eliv ery and expression, w hich can then be p ro d u c e d in significant q u an tities re la tiv e ly easily in a bacterial fe rm en tatio n .

Figure 2.2. DNA Conform ation (a) The F or fertility fa cto r o f E.coli represents 1-2% o f its total

cellular DNA and is an example o f a plasmid. Plasmids are se lf replicating, circular double stranded (ds) pieces o f DNA found distinct from the main bacterial chromosome, (b) Open Circular DNA (ocDNA) can exist in relaxed or (c) Super Coiled (scDNA) form, topoisomers, which is more compact, (d) If circular or scDNA form s are nicked they become linear DNA (Fishman and Patterson, 1995).

(b)

C irc u la r D N A

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L in ea r D N A S u p e rc o ile d D N A

Claire Nicole Mount

Chapter!.____________________________________________________________________Literature Survey