Estrategia 1

In order to improve the efficiency and circumvent the problems associated with the low efficiency of gene delivery vectors many avenues of research are being explored and strategies being developed to increase the bioavailability to specific populations of cells are of particular interest as they offers two distinct advantages. A strategy which increases bioavailability of gene delivery vectors not only provides the ability to target vectors to specific cells, thereby increasing the efficiency, but also offers a way of prolonging circulation time by avoiding removal by circulating and fixed components of the host immune system (Hug and Sleight, 1991; Kaneda, 2000). There are three complementary strategies used to increase the bioavailability of synthetic vectors including changing the route, changing the composition of the liposomes and finally adding targeting molecules to the surface of the lipids (Hug and Sleight, 1991).

The incorporation of targeting ligands during formulation to produce vectors that target and transfect specific cells and tissues has recently become a popular strategy seen as a major step towards effective therapeutic gene transfer (Chonn and Cullis, 1998; Dalluge et a i , 2002; Morpurgo et a i, 2002; Parkes and Hart, 2000). M any novel DNA complexes consist of a lipid, protein, peptide or polymeric carrier plus ligands to promote recognition and uptake by specific tissues (BBSRC business, July 2000; Davis, 2002; Dalluge et a l, 2002). The ligands used are added by ionic interactions or covalent attachments and are capable of targeting the DNA complex to the cell surface receptors on the target cells, promoting cell specific binding (Figure 2.3) (Hart et a l,

1998; Ledley, 1995; Uherek and Weis, 2000). If this strategy is optimised it offers a way to increase the efficiency of the drugs as the therapeutic agent will go directly to where it is required thereby reducing the doses needed for a complete treatment

Chapter!.____________________________________________________________________Literature Survey

(Smyth Templeton and Lasic, 1999). Targeting can also improve the therapeutic index of gene transfer by preventing damage of healthy tissue and decreasing the risk of germ line transduction (Galanis et al., 2001). The attention on targeted delivery has resulted in a vast number of ligands being identified as potential targeting agents most of which are elements of the bodies own targeting mechanisms. The decision of which targeting method to use depends on a detailed knowledge o f the target cells and their microenvironment (Hug and Sleight, 1991). At present research is being carried out on the use of cell receptor ligands such as transferrin and folate, peptides, viral coat proteins and antibody fragments (Choi et at., 1999; Iden and Allen, 2001; Lee et at.,

1997; Lee and Huang; 1996; Parkes and Hart, 2000; Zauner et at., 1998).

The identification and use of natural peptides has allowed the development of synthetic gene delivery systems that mimic the entry o f pathogens into the cell. The sequence of synthetic peptides can be derived from known proteins or can be engineered de novo

on the basis of known structural and chemical requirements for the desired property. Alternatively new sequences with useful characteristics can be identified by screening random peptide libraries, such as phage display libraries (M orpurgo et at., 2002). Advances in the use of targeted peptide/DNA vectors are emerging from the isolation of peptides that are, not only capable of binding the cell surface ligands with high affinity but also have the ability to efficiently induce internalisation to the interior of the cell nucleus (Morpurgo et a l, 2002; Parkes and Hart, 2000).

Integiins, the family of heterodimeric membrane proteins, are just one example o f the type o f peptides currently utilised in gene delivery as binding ligands and have been studied and used by various research teams (Parkes and Hart, 2000). Integiins are found on all cells, including airway fibroblasts, epithelial cells and myeloid cell lines, and play important physiological roles in the attachment o f cells to extracellular matrix, cell-cell interactions, cell migration, apoptosis and in signal transduction (Hart, 1999; Jenkins et a l, 2000). However, binding of cell surface integiins is also exploited by a number of pathogenic organisms including Y. pseudotubercuolosis and adenovirus to promote cellular internalisation (Hart, 1999; Jenkins et a l , 2000). This mechanism of cell entry has been exploited in the development o f a novel family of

Chapter!.____________________________________________________________________Literature Survey

integrin targeted non-viral vectors (Jenkins et a l , 2000). Peptide DNA complexes, consisting of an integrin targeting molecule with a polylysine DNA binding domain have been shown to be capable of mediating delivery of genes to many cell types, including epithelial, endothelial and melanoma cells, in an integrin dependant manner (Parkes and Hart, 2000).

Cell surface integrin receptors offer a number of advantages for the development of efficient targeted viral and synthetic delivery systems (Hart, 1999). For example, integrin targeting has the advantage that it mediates both efficient cell attachment and subsequent cell entry (Hart, 1999). Integrin binding peptide ligands are usually short as few as six amino acid residues which permits then to be displayed at appropriate sites on the viral capsid proteins, such as the fiber of adenoviruses, by genetic engineering of the viral genome and also means they have a low potential immunogenicity (Hart et

a l , 1995; Hart, 1999). Furthermore, the small size o f these peptides means that they

can be easily synthesized chemically to a high degree o f purity and can be easily incorporated into electrostatic non-viral vector complexes. Integrin mediated internalisation is also reportedly less limited by particle size than other forms of receptor mediated endocytosis. As a result greater amounts of DNA may enter the cell and it should, theoretically, be possible to deliver larger constructs up to sizes that have been proposed for human artificial chromosomes (Hart et a l , 1995). Finally, some peptide ligands bind to a range of integrins displayed on many cell types while others are more restricted in their receptor affinities and so can be selected as appropriate to the cellular target (Hart, 1999).

Jenkins and co-workers (2000) reported the use of an integrin-targeted non-viral gene delivery system for pulmonary gene transfer (Jenkins et a l , 2000). These vectors consist of a cationic liposome (L), an integrin binding peptide (I) with a sixteen-lysine tail and plasmid DNA (D) that combine electorstatically to form the LID vector complex (Figure 2.6). They demonstrated that this vector can deliver the genes to the lung, transfecting both bronchial epithelium and parenchymal cells with similar efficiency to an adenoviral vector and with greater efficiency than a cationic liposome (Jenkins et a l, 2000). Furthermore, it was shown that the vector may be administered

Chapter2. Literature Survey

rep eated ly , lead in g to fu rth e r g ene ex p ressio n , w ith o u t in d u c in g in fla m m a tio n (Jen k in s et a l , 2000). T h e featu res o f the L ID com p lex p ro v id e ad v a n ta g e s o v e r cu rren tly av a ila b le vecto rs, w hich co u ld p ro v e b eneficial in the tre a tm e n t o f d isease s such as

cy stic fib ro sis, asth m a, p u lm o n ary fib ro sis and n o n -sm all cell lung c a n c e r (Je n k in s et

a i , 2000).

Figure 2.6. Formation o f Lipopolypeptide Particles comprising lipofectin (L),

cyclic integrin-binding peptide (I) and plasmid DNA (D).

i

C yclic in teg rin -b in d in g

p ep tid e (I) L ip o fec tin (L )

P lasm id D N A (D)

V

L ID C o m plex

A dapted from Hart, 1999.

Claire Nicole Mount

Chapter!. Literature Survey

R eco m b in an t an tib o d ies, h av in g m ad e a co m eb a ck to th e ra p e u tic clin ical ap p lica tio n s, have also fo u n d a role in g en e th e ra p y as targ e tin g lig an d s (H o llig e r an d H o o g e n b o o m ,

1998; N ielsen et a i , 2002; T sai et a i , 1998). S uch lig an d s are ideal targ e tin g

c o m p o n en ts fo r n o n -v ira l d eliv ery sy stem s as th ey are n o n -to x ic, ea sily sy n th esised , w ell d e fin e d seq u e n ces and are ea sily purified. It is also p o ssib le to en g in e e r the an tib o d ies so that th ey h o ld the d e sire d p ro p erties in ju s t a frag m en t o f th e an tib o d y ra th e r than the w h o le stru ctu re (F ig u re 2.7) (F ilp u la and M cG u ire, 1999; H o llig er and H o o g e n b o o m , 1998; H u d so n , 1998). T h e ability to b u ild larg e phage an tib o d y libraries o f c o m b in atio n s o f hu m an heav y and light ch ain g en es has recen tly b een d ev e lo p e d and has a llo w ed the isolatio n o f hu m an an tib o d ies to v irtu ally any an tig en in clu d in g self an tig en s (Ja g er and P lu c k th u n , 1999; Jo n e s an d M arasco , 1998). T h e an tib o d y frag m en ts are c o n ju g a te d to the lipidic v ectors via lin k e r m o lecu les to pro d u ce ‘im m u n o lip o so m e s’ that have the ca p acity to re co g n ise the an tig en s on targ e ted cells

th ereb y re su ltin g in in cre ased e ffic ie n c y o f d eliv ery (N ielsen et a l , 2002; T sai et a l ,

1998).

Figure 2.7. Antibody Fragments

A ntigen B in d in g D o m ain

r--- A

L IG H T C H A IN L IG H T C H A IN •raem en l F r a g m e n t H E A V Y C H A IN W h o l e Anti hodV S cF v (Vh+ Vl) F ragm ent Disulphide bond H E A V Y C H A IN

Key: V - Variable Domains

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Finally the design of a gene delivery system designed to incorporate the best features both liposomal and recombinant viral approaches has provided a new and promising field of drug delivery (Davies et a i, 1998; Geddes and Alton, 1995). As a hybrid between virus and liposome, referred to as a virosome, the novel vectors will have superior efficiency (Figure 2.8) (Bally et al., 1999; Kaneda, 2000). Viral functioning is conferred on liposomes by direct coupling of viral proteins or viral envelopes to liposomes producing vectors that possess properties for efficient fusogenic delivery and tissue tropism derived from viral molecules (Uherek and W eis, 2000; Kaneda, 2000). These properties will enable receptor-mediated endocytosis, escape from the endosome and potentially nuclear uptake in a manner analogous to viruses, plus lessened toxicity derived from the use of a liposome element (Pouton et a l, 1998). For example. Plank and co workers showed that fragments o f the haemagglutinin protein from the influenza virus could not only mediate pH-dependant lysis of liposomes and erythrocytes but could also increase the frequency o f transfection when incorporated into DNA/PLL complexes, provided there is a high cationic charge ratio (Pouton and Seymour, 1998). Other advantages of this novel technology include their efficiency, in terms of the short incubation time which reduces the potential for degradation of DNA in the endosome and lysosome thereby making them useful both in vitro and in vivo

(Kaneda, 2000). The major draw back to the use of these novel vectors is the possibility that they may elict an immune response due to their viral elements (Bally et

a l , 1999). However research is ongoing and at present a num ber o f viral proteins are

well characterised and if more viral membrane proteins or whole viral envelopes can be isolated and reconstituted with lipids the development of numerous hybrid vectors with varied tissue targeting specificities, high efficiency and low toxicity will be possible (Kaneda, 2000).

Although, in theory, viral vectors can be re-targeted, these alterations are time consuming and complex in practice. In comparison, production o f targeted synthetic vectors, or stealth vectors, is relatively trivial (Parkes and Hart, 2000). With the use of advances in phage display technology it will become easier to isolate receptors that can mediate internalisation of the vector complexes to a variety o f tissues and cell types in an efficient and specific manner (Parkes and Hart, 2000). Compared to the viral

Chapter2._________________________________________________________________Literature Survey

system , stealth v ectors also have the ad v a n ta g e o f b e in g n o n -p a th o g e n ic an d are able to

p ack ag e a m uch la rg e r q u an tity o f nucleic ac id in th e co m p lex (H art et a l , 1998).

T h ere are a n u m b e r o f param eters that re m a in to be o p tim ise d befo re it is p o ssib le to achieve th e u ltim ate goal o f fo rm in g h o m o g en eo u s, sm all, so lu b le an d stab le n o n-viral D N A p artic le s w ith high transfection efficien cy . R e c e p to r m e d ia te d g en e d eliv ery is

h o w e v er b e c o m in g the m ost pro m isin g ap p ro ach (L ed ley , 1995; T sai et a l , 1998;

Z a u n e r et a l , 1998).

Figure 2.8 - Virosome Construction

Q Q O

V

L ip id s P la sm id D N A

O

D N A loaded liposom e ^ | ^ I V iral P ro te in s OR V iral e n v e lo p e D N A lo aded virosom e

ChaDter2.____________________________________________________________________Literature Survey

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