POLÍTICA MONETARIA

The antihaemophilic FVIII :C is a trace plasma glycoprotein which plays a central role in normal blood coagulation. FVIII:C circulates as a pro-cofactor bound to von Willebrand factor (vWF) which protects it against cleavage by factor IXa or protein C. FVIII is activated

by cleavage by either throm bin or less so by activated factor X (Xa). In this process it is released as FV IIIa from vW F and acts now as a cofactor o f activated factor IX for the activation of factor X in the presence of calcium ions and phospholipids. FVIIIa is inactivated by activated protein C.

The mature full length factor VIII protein is com posed o f 2332 amino acids and has a m olecular weight o f 330,000-360,000 (W ood et al., 1984; Rotblat et al., 1985), depending on the extent o f post-synthetic glycosylation. The protein can be divided into distinct dom ains o f hom ologies and/or function. The arrangem ent o f the triplicated A-dom ains (A I, A2, A3),

the large B-domain (aa nos. 712-1648, W ood et al., 1984) and the two C-dom ains is

schem atically shown in Fig. 1.3.1. (Vehar , 1984). Deletion experim ents have shown that

most o f the B-domain is dispensable for in vitro procoagulant activity (Toole et al. 1986a and 1986b; Burke et al., 1986). This domain, unlike the A and C dom ains, does not share any sequence homologies with any other protein. It does not have any known function in itself and is proteolytically cleaved from FVIII during activation by throm bin after amino acids nos 372 (R/S) and 1689 (R/S) (Eaton et al., 1986).

A1 A2 B A3 C1 C2

a1

a2

Fig. 1.3.1: Schematic representation of the FVIII protein domain structure. T he dom ain structure o f the FVIII protein is A 1 -A 2 -B -A 3 -C I-C 2 (Vehar e t a l., 1 984), w ith the acidic peptides a l and a2 found in the jo in in g region betw een dom ains A I -A 2 and B -A 3. The dom ains are com prised o f the fo llo w in g am ino acids; A l :

1 -336, a l: 3 3 7 -3 7 4 , A2: 3 7 5 -7 1 9 , B: 7 2 0 -1 6 4 8 , a2: 1 6 4 9 -1 6 8 9 , A 3: 1 6 9 0 -2 0 1 9 , C l: 2 0 2 0 -2 1 7 2 , C2: 2 1 7 3 -2 3 3 2

(quoted in Tuddenham and C ooper, 1994).

The chromosomal locus o f the FVIII gene has been established as Xq28. The gene itself is comprised o f 186 kb and consists o f 26 exons, the largest o f which is exon 14 with 3106 bp, containing the whole B-domain (Gitschier et al. , 1984), The mRNA o f 9009 bp (Toole et al. , 1984) contains large untranslated regions (150 bp 5'UTR and 1806 bp 3'UTR), a 47 nucleotide sequence coding for a 19-amino-acid signal peptide and a 6996 nucleotide sequence encoding the 2332 amino acid protein.

1.4.

Research aims

In this introduction the structure and function of muscle and its proteins has been outlined with an emphasis on myosin, the major component o f the contractile apparatus. The existence o f at least 11 muscle MyHC isoforms and their tight regulation in a fibre type-specific and developmental stage-specific manner was described in the context o f transcriptional regulation. The importance o f muscle-specific promoter and enhancer regulatory elements for the expression o f particular genes was linked to their potential use in a gene therapy approach with muscle as an in vivo expression system for introduced exogenous genes. The feasibility o f using intramuscular direct gene injection as a form o f gene therapy for haemophilia A was presented.

At the start o f this work only two regulatory sequences o f the different members o f the MyHC gene family were known. Thus, in summary, the major aims o f this research project were:

(I) To isolate, identify and characterize different isogenes o f the MyHC multigene family in rabbit

(II) To identify potential promoter elements and to characterize them by sequencing and expression studies

(III) To design a strong muscle specific expression cassette for the use o f direct gene injection into muscle containing muscle specific promoter and enhancer elements (IV) To test this cassette in vitro for the expression o f the factor VIII gene as a step

towards an effective form o f gene therapy for haemophilia A.

A graphical outline o f the research strategy is given in Fig. 1.4.1.

o

Isolation and characterization of MyHC isogenes

genomic clones

Identification and characterization of potential regulatory elements

\ /

FVIII

I

In vitro transfection of myoblasts

\ /

Designing a musde-spedfic expression cassette containing MyHC and MyLC regulatory

elements and the FVIII gene

Test for FVIII expression in vitro in muscle cell cultures

\ /

R e a d y t o b e t e s t e d in vivo b y d i r e c t g e n e I n je c tio n

Fig. 1.4.1: Flow-diagram of Research Aims. For details see text.

Chapter 2. Materials and Methods

2.01. Materials

2 .0 1 .1 . Standard reagents

All reagents were analar grade or equivalent and obtained from BDH, Sigma and Fisons unless stated otherwise.

2 .0 1 .2 . Electrophoresis reagents

Acrylamide was obtained from Sigma and Flowgene, TEMED and Sigmacoat from Sigma, agarose from Sigma and urea from BDH

2 .0 1 .3 . Enzymes

Enzymes were obtained from the following sources: DNAse (Boehringer), Klenow (Boehringer, Promega), T4 Ligase (Boehringer, Promega, Stratagene), Mung bean nuclease (Sratagene, Boehringer), alkaline Phosphatase (Boehringer), Proteinase K (Promega; dissolved to 10 mg/ml HjO, aliquotted and stored at -20°C), Restriction enzymes (Boehringer, NBL, Promega, Gibco-BRL, Stratagene), Reverse transcriptase (Boehringer), RNAse (Boehringer; lOmg/ml were dissolved in lOmM Tris-HCl, pH 7.5 , and 15mM NaCl. To denature the D N Ase, the tube was placed in boiling water for 15 min, cooled to room temperature, aliquotted into Eppendorfs and stored at -20°C.), DNAse free RNAse (Qiagen), Taq DNA polymerase (Boehringer, bioline, Promega, Cetus, Gibco-BRL), T4 DN A polymerase (Boehringer).

2 .0 1 .4 . Molecular weight markers

The following weight markers were used: RNA ladder (9.5kb - 0.24kb) from Gibco-BRL; Lambda DNA from Promega; Lambda H indlll from Promega; Lambda H indW llE cd^ from Promega; Lambda Pstl was self-made from lambda DNA; DNA Ikb ladder was the size marker X from Boehringer Mannheim and pBR Hinfl was self-made from pBR 322 DNA

2 .0 1 .5 . Bacterial strains, Vectors, DNA-Libraries and Clones A detailed listing is given in Appendix E .l.

2 .0 1 .6 . Mammalian Cell Lines

Specifications o f the mammalian cell lines used are listed in Appendix E.2.

2 .0 1 .7 . Radiochemicals

^^S-a-dATP, ^^P-a-dCTP and y-dATP from Amersham and ‘'‘C-Chloramphenicol from NEN (New England Nuclear).

2 .0 1 .8 . Miscellaneous

Sterile universals, bijoux, falcon tubes, pipettes, petri dishes, tissue culture dishes and cell culture flasks obtained from Greiner; Nylon membranes (Hybond-N and Hybond N + ) and nitrocellulose filters (Hybond-C Extra, 0.45 micron, 82mm) from Amersham; X-ray films from Fuji (GRI) and Amersham; yeast tRNA from Sigma; lyophilized herring sperm DNA from Boehringer; Sephadex G50 from Pharmacia; yeast extract from Merck; agar from Oxoid and phenol from Amresco

2 .0 1 .9 . Common Buffers

lOx Loading buffer (20% Ficoll 400, 1 % SDS, 0. IM NajEDTA, pH 8, 0.25% Bromophenol Blue, 0.25% Xylene Cyanol (optional, runs 3-4x slower than BpB)

50x TAE buffer (L '): 242g Tris-HCl base, 57.1ml glacial acetic acid, 0.05 M EDTA, pH8 lOx TBE buffer (L ‘): 108g Tris-HCl base, 55g boric acid, 40ml 0.5 M EDTA, pH 8 lOOx Denhardt's: 2% BSA, 2% Ficoll, 2% PVP (polyvinyl pyrrolidone)[w/v]

NaCl/EDTA: 75mM NaCl, 25mM EDTA, pH 8.0

Phenol-Chloroform: A 1:1 v/v mixture o f equilibrated phenol and chloroform; isoamyl alcohol (24:1 v/v), stored under TE (pH 7.8) at 4°C.

SDS: Sodium dodecyl sulfate (sodium lauryl sulfate). Stock solutions o f 10% and 20% were prepared in sterile water and adjusted to a pH o f 7.2 with concentrated HCl. Dissolution was facilitated by heating to 68°C.

20x SSC: 3M NaCl, 0.3M Na-citrate, adjusted to pH 7.0 with ION NaOH TE: lOmM Tris-HCl, ImM EDTA, pH 7 .5 , 8.0 and 9.0

TENS: lOmM Tris, pH 7.5, ImM EDTA, pH 7.8, O.IM NaOH, 0.5% SDS TNES: 0.14M NaCl, 20mM Tris-HCl (pH 7.6), 5mM EDTA (pH 8.0), 0.1% SDS

2 .01.10. Microbiology media:

2xYT media (L ‘): 16.Og Tryptone, lOg NaCl, 5g Yeast extract

LB (Luria-Bertini) media (L ‘): lOg Tryptone, lOg NaCl, 5g Yeast extract

Agar plates made by adding 7.5g Bacto agar to 500ml of the above media. Antibiotics were added as required once the media had cooled to 50°C.

Top agarose overlays: media as above with 0.7% agarose

SM (L ‘): 5.8g NaCl, 2g MgS0 4-7H2 0 , 50ml IM Tris-HCl, pH 7.5, 5ml 2% gelatin solution

2.02-15. Methods

All solutions and glassware for nucleic acid work were sterilized by autoclaving at 15 p .s.i., 120°C for 20 min. Heat labile solutions were sterilized by filtration through 0.22 /xm pore sized filters. Disposable gloves were worn for all experimentations. The basis for most o f the methods used in this thesis can be found in Sambrook et a/.(1989) and in Current Protocols'.

2.02. DNA Preparation

2 .0 2 .1 . Plasmid DNA preparation

2 .0 2 .1 .1 . Lai^e scale. 5ml growth medium (LB or 2xYT with appropriate antibiotics) were inoculated with a bacteria/plasmid colony from an agar plate or with 10/xl o f a glycerol stock and grown for 5 to 8 hours at 37°C, 250 rpm. 250-500ml medium with appropriate antibiotics were inoculated with the 5 ml culture and incubated in a 37°C shaker at 200-250 rpm for 12- 16 hours. The cells were pelleted for 15 min at 4°C at 4000 rpm in a Sorvall GS3 rotor. The cell pellet was either washed with 100ml TE, pH 8.0, and stored at -20°C or processed immediately. Plasmid isolation and purification were originally carried out by the CsCl method. This method was omitted when high yield and quality o f plasmid DN A were obtained by the use o f Qiagen colunms (supplied by Hybaid). For details o f method see manufacturer's protocol. DNA was precipitated with 0.7 volumes o f isopropanol, pelleted at 30,000 g and was washed in 70% ethanol. The pellet was air-dried at room temperature and the D N A was taken up in 200-300/xl sterile water or TE and stored at 4°C for short term use or at -20°C as a stock.

2 .0 2 .1 .2 . Small scale. 5ml growth medium (LB with appropriate antibiotics) were inoculated with a bacteria/plasmid colony from an agar plate or with 10/xl from a glycerol stock and grown for 12 16 hours at 37°C, 250 rpm. Alternatively 3ml were grown for 5 hours or 1ml was grown directly in a 2ml Eppendorf tube, placing the tube vertically in the shaker (250 rpm) for several hours. All centrifugations mentioned below were carried out in bench-top micro-centrifuges (MSB Micro Centaur).

2 .0 2 .1 .2 .1 . Purified plasmid DNA mini preparation. Adapted after Birnboim and Doly (1979) and Ish-Horowitz and Burke (1981). A small scale alkaline lysis method was carried out. I.5m l o f liquid culture were spun in an Eppendorf at 5,000 rpm for 5 min at RT. The cell pellet was resuspended in 100^1 solution MIP-I (10% glucose, lOmM EDTA, 25mM Tris- HCl, pH 8.0) by vortexing and incubated at RT for 5 min. 200/^1 MIP-II (200mM NaOH, 1 % SDS) were added, mixed well and incubated on ice for 5 min. 150^1 MIP-lII (3M KO Ac, II.5% acetic acid) was added, mixed immediately and placed on ice for 15 min. The tubes were spun in an Eppendorf centrifuge at 13,000 rpm for 10 min and the supernatants transferred to new Eppendorf tubes. 400/xl Phenol:chloroform were added and mixed well to extract the DNA. After centrifugation at 13,000 rpm for 5 min the upper layer was transferred to a new Eppendorf. The DNA was precipitated by the addition o f 40fil 3M NaOAc (pH 5.2) and 900/ri absolute ethanol and incubating at -70°C for 30 min. The DNA was pelleted at 13,000 rpm for 20 min, washed in ice-cold 70% ethanol, dried at 37°C and dissolved in 50/^1 sterile H^O. With this method 2 to 3/xl o f the DNA preparation were sufficient for a restriction digest.

2 .0 2 .1 .2 .2 . Rapid plasmid mini preparation. A 1ml culture (in 2ml Eppendorf tube) was spun at 13,000 rpm for 10 sec. The supernatant was tipped o ff with one shake, leaving 50 to 100/d LB remaining. The pellet was re suspended by high speed vortexing, 300 fi\ TENS were added and the suspension vortexed for 2-5 sec. The tubes were placed on ice if more than 10 min were needed before next step. \50fi\ 3M NaOAc, pH 5.2, was added and vortexed for 2-5 sec. After a 2 min centrifugation at 13,000 rpm the supernatant was transferred to a fresh Eppendorf tube, 900/xl absolute ethanol (-20°C) was applied, mixed, and centrifuged again at 13,000 rpm for 6 min. The pellet was washed 2x with ice-cold 70% ethanol, dried for 5-10 min at 37°C and resuspend in either 100/d TE (pH 8) or sterile water. For restriction digests 10/xl were used when looking for small inserts between 100-500 bp or 5/xl for large inserts (1000 bp and more).

2.0 2 .2 . Bacteriophage DNA preparation

2 .0 2 .2 .1 . Infection o f host cells with phages. For each phage infection a fresh culture of E .coli cells (LE392 cells for Charon 4A and NM539 cells for EMBL-3) was prepared. Cells were grown for 12 to 16 hr in 5ml LB supplemented with lOmM MgSO^ (50/il o f IM stock) and 0.2% maltose (50/il o f 20% stock). Host cells and bacteriophage were mixed and incubated at RT for 15-30 min. For different applications the ratio o f host cells to pfu of phage varied as detailed further on.

2 .0 2 .2 .2 . Titrating phages. A dilution series o f phage lysate supernatant (10^ to 10 '°) was prepared in SM. 3ml molten top agarose (0.7%) were placed into autoclaved test tubes and left to stand in a water bath at 47°C. 100/d host cells were mixed with lOO/il phage for each dilution and incubated at RT for 15 min. The following step was carried out for each host cell phage mix separately; the mix was added to a tube with top agarose, gently vortexed and immediately poured onto a prewarmed 90mm LB/agar plate (37°C). Once the top agarose was set, the plates were inverted ana incubated for 5-8 hr at 37°C. The number o f plaques per plate were counted to determine the plaque forming units (pfu) per ml o f the original lysate.

2 .0 2 .2 .3 . Growing phages on culture plates. For screening lambda phage libraries 200/tl E .coli were infected with 10^ pfu for 90mm dishes and 3x10^ pfu for 150mm dishes. The latter was carried out with 10ml molten top agarose. The plates were incubated at 37°C until a bacterial lawn had grown with plaques ranging between 0.25m m and 0.5m m. The plates were then chilled at 4°C to allow the top agarose to harden. If not processed immediately the plates were stored at 4°C. Plaque lifts onto nylon membranes is described in section 2 .0 5 .3 .2 . and hybridization with DNA probes in section 2.05.5.

2 .0 2 .2 .4 . Isolation o f individual phage colonies. To isolate a plaque from a plate the wide end o f freshly autoclaved yellow tips (for 0.2ml) or Pasteur-pipettes were used to stab through the plaque into the hard agar beneath. The agar and plaque were lifted, if necessary with mild suction, and placed into a polypropylene tube with 1ml SM and a drop o f chloroform. The tubes were left to stand for 1-2 hr at RT so phage particles could diffuse into the SM. After centrifugation for 10 min at 13,000 rpm the supernatant was either stored longterm at 4°C with a drop o f chloroform or used to infect fresh host cultures to produce a higher titre lysate. One plaque commonly yielded 10^-10^ pfu.

2 .0 2 .2 .5 . Preparation o f high titre lysate stocks. High titre lysates were prepared from liquid cultures as well as from plate cultures (90mm dishes). A high infection o f 100^1 host cell culture with 5x10^ pfu was chosen for the preparation o f plate lysate stocks. Infection and plating was carried out as detailed above. The plates were incubated at 37°C for 8 to 16 hours

until the plaques touched each other. Plates were briefly chilled at 4°C, 3ml SM were added and left to stand at RT with intermittent, gentle shaking for 2-3 hours. The SM with the diffused phage particles was then transferred into 15ml polypropylene tubes and lOO/xl chloroform were added. The plate was incubated with a further 2ml SM for 20-30 min which was then pooled with the first harvest. The lysate was briefly vortexed and centrifuged at 4000g for 10 min at 4°C. The supernatant was mixed with a drop o f chloroform and stored as a phage stock at 4°C. The titer was determined as outlined in section 2 .0 2 .2 .2 .

2 .0 2 .2 .6 . Phage DNA preparation from liquid culture. For each phage DN A preparation a 5ml host cell culture was infected with 2.5 x 10^-10® pfii, incubated for 15 min at RT and added to a 11 flask with 250ml LB, containing lOmM MgSO^. Incubation at 37°C at 300-350 rpm was carried out until lysis occurred. 1ml chloroform was added and the flask left to

shake a further 10-15 min to ensure complete lysis o f the bacteria. After centrifugation for 10 min at 10,000 rpm in a JIO Beckman fixed angle rotor or for 15 min at 5,000 rpm the supernatant was transferred into a fresh flask or centrifuge bottle. (A 1.5ml aliquot was taken at this stage and stored in a 2.5m l screw cap tube with a drop o f chloroform at 4°C as a fresh phage stock). If the lysate was not processed further the same day, 1ml chloroform was added and the flask stored at 4°C. The supernatant was then centrifuged again before the next step. Phage precipitation and DNA purification was carried out with Qiagen columns (Hybaid) according to manufacturers instructions. In brief, the supernatant was incubated at 37®C for 30 min with 400/il solution LI (20mg/ml RNAse, 6mg/ml DNAse I, lOOmM Tris-HCl (pH

7.5), lOmM EDTA, 300mM NaCl, 200/ig/ml BSA, pH 7.5). 50ml 30% PEG/ 3M NaCl was added and the samples incubated on ice for 1 hr. After centrifugation at 10,000g for 10 min the pellet was re suspended in 9ml solution L3 (lOOmM Tris-HCl, lOOmM NaCl, 25mM EDTA, pH 7.5). 9ml 4% SDS was added and the samples incubated at 70°C for 20 min, then cooled on ice. After addition o f 9ml 2.55M KOAc (pH 4.8) the lysate was immediately but gently mixed and centrifuged at 15,000g for 30 min at 4°C. If the supernatant was not clear a second identical spin was performed for 15 min. The supernatant was passed through an equilibrated Qiagen column, the column was washed and the DNA eluted according to manufacturers recommendations. The DN A was precipitated with 0.8 vol isopropanol.

pelleted by centrifugation, washed with 70% ethanol and briefly dried at RT. The DN A was dissolved in 300-400/iil H2O and stored at 4®C.

2 .0 2 .3 . Genomic DNA

2 .0 2 .3 .1 . Isolation o f DNA from whole blood. Blood was taken up in EDTA or citrate (3.8% trisodium citrate used in a ratio o f 1:9 anticoagulant to blood) and frozen in 50ml falcon tubes. The samples were thawed at RT and sterile distilled HjO was added up to 45ml. 4ml o f 20% Triton-X-100 was added, mixed and centrifuged at 2000g and 4°C for 20 min. Samples were placed on ice, the supernatant discarded and the excess removed with a pipette. The pellet was resuspend in 2.5ml NaCl/EDTA (75 mM/25 mM, pH 8), 200/rl 10% SDS

were added and mixed by inverting the tube. lOOfil Proteinase K (10 mg/ml stock) were