Prenatal Gene Therapy || Choice of Surrogate and Physiological Markers for Prenatal Gene Therapy

273

Charles Coutelle and Simon N. Waddington (eds.), Prenatal Gene Therapy: Concepts, Methods, and Protocols, Methods in Molecular Biology, vol. 891, DOI 10.1007/978-1-61779-873-3_13, © Springer Science+Business Media, LLC 2012

Chapter 13

Choice of Surrogate and Physiological Markers for Prenatal Gene Therapy

Juliette M.K.M. Delhove , Ahad A. Rahim , Tristan R. McKay , Simon N. Waddington , and Suzanne M.K. Buckley

Abstract

Surrogate genetically encoded markers have been utilized in order to analyze gene transfer ef fi cacy, location, and persistence. These marker genes have greatly accelerated the development of gene transfer vectors for the ultimate application of gene therapy using therapeutic genes. They have also been used in many other applications, such as gene marking in order to study developmental cell lineages, to track cell migration, and to study tumor growth and metastasis. This chapter aims to describe the analysis of several commonly used marker genes: green fl uorescent protein (GFP), β -galactosidase, fi re fl y luciferase, human factor IX, and alkaline phosphatase. The merits and disadvantages of each are brie fl y discussed. In addition a few short examples are provided for continual and endpoint analysis in different disease models including hemophilia, cystic fi brosis, ornithine transcarbamylase de fi ciency and Gaucher disease.

Key words: Green fl uorescent protein , GFP , Luciferase , β-galactosidase , Bioluminescence , Alkaline phosphatase , Quantitative PCR , Immunohistochemistry

Gene therapy of monogenic diseases is perfected by achieving tissue-targeted expression resulting in appropriate levels of gene expression whilst using the smallest amount of gene transfer vector possible. Moreover, the gene expression is ideally regulated in a physiologically appropriate fashion. Although other gene therapy targets, such as cancer, depend upon expression of genes which may be toxic and which may be expressed in extremely non-physi-ological concentrations, the concept of expression targeting and regulation remains valid. To interrogate the fundamental mecha-nisms of vector function and as a precursor to application of thera-peutic transgenes, surrogate marker genes are widely used. These have been used extensively in pre and postnatal gene transfer and

1. Introduction

274 J.M.K.M. Delhove et al.

gene therapy in order to analyze the ef fi cacy, duration, and location of gene expression following administration of gene transfer vectors. In addition, marker genes may also permit monitoring of transduced cell lineages (ideally if the marker gene has integrated into the cell genome) and to monitor tumor growth and metastasis. More recently, marker genes have been used to monitor intracellular sig-naling pathways and have been placed downstream of known regu-latory sequences (including promoters and enhancers) in order to act as surrogate markers of endogenous gene expression.

One of the fi rst examples in preclinical studies was the delivery of the neomycin-resistance gene (neo R ) into blood and bone mar-row cells. The phosphotransferase activity of the gene product was determined in tissue extracts ( 1 ) . Since then, several other enzymes have been used as genetically-encoded marker proteins, including human α -1-antitrypsin ( 2 ) and chloramphenicol acetyltransferase, although β -galactosidase ( β -Gal) and human placental secreted alkaline phosphatase (SEAP) ( 3, 4 ) have been more widely used (and are thus described in this chapter). β -Galactosidase can be assayed in tissue homogenates by several means but one of the preferable assays is by ELISA. It can also be located by histochemi-cal staining of whole or sectioned tissues using a substrate which forms a blue precipitate after reaction with the enzyme ( 5 ) . SEAP, being a secreted enzyme, can be measured in plasma and also in tissue culture medium. Similarly, human factor IX (hFIX) can be detected in plasma and is particularly useful for long-term, non-invasive expression studies. It can also be detected by immunohis-tochemistry in tissues ( 6 ) .

Green fl uorescent protein (GFP) is derived from the jelly fi sh Aequorea victoria ( 7 ) . It emits a green light when excited by light in the long wave ultraviolet region of the spectrum. It can be visu-alized directly in whole tissues, can be detected by immunohis-tochemistry, and can be quanti fi ed by ELISA in tissue homogenates. Subsequently, several homologs of jelly fi sh GFP have been devel-oped which emit light at different wavelengths, such as blue, yel-low and red fl uorescent proteins. More recently, fl uorescent proteins from other organisms, such as the sea anemone Entacmaea quadricolor , have been identi fi ed and are subject to further modi fi cations to improve their brightness and stability. Moreover, it is now feasible to detect these proteins by whole-body imaging and the ef fi ciency of this technology has been improved by modi fi cation of these fl uorescent proteins so that they emit light towards the infrared end of the spectrum as light at these wave-lengths is least absorbed by mammalian tissue. Over the past 10 years, whole-body imaging has been revolutionized by the use of the enzyme luciferase from the fi re fl y Photinus pyralis ( 8, 9 ) . This enzyme converts D -luciferin into oxyluciferin, consuming ATP and oxygen in the process but, most importantly, also emit-ting light ( 10 ) . This light is emitted at 557 nm and is therefore

27513 Choice of Surrogate and Physiological Markers for Prenatal Gene Therapy

yellow-green in color, however, red-shifted variants have been developed which are absorbed less by mammalian tissue and are, therefore, more suited for whole-body bioimaging ( 11 ) .

Each marker gene has advantages and disadvantages, therefore, the use of more than one marker gene should be considered. For example, there is some evidence that GFP is toxic in certain cell types ( 12 ) and that expression levels of different colored fl uorescent proteins depend upon the tissue type ( 13 ) . Secreted marker genes such as SEAP or hFIX are particularly useful for long-term, nonin-vasive, and nonterminal monitoring of gene expression, however, ef fi ciency of release may depend upon the cell type which has been transduced and therefore may not be the same for all tissues. Luciferase has proven immensely useful of late for performance of whole-body bioluminescence and can, therefore, be used for lon-gitudinal monitoring and localization of gene expression. However, immunohistochemical detection of luciferase is poor; nevertheless, this has been overcome by the addition of a peptide tag (e.g. FLAG, his, myc), generation of a GFP fusion construct or incorporation of GFP in a bicistronic cassette.

Physiological endpoints indicating therapeutic success of gene therapy will naturally vary from disease to disease. For many pre-clinical gene therapy studies, survival and weight gain/loss would be useful generic markers. However, most of the longitudinal and endpoint analyses in these cases will very speci fi cally depend on the symptoms of the disease being studied and details of their determi-nation would be beyond the scope of this book. To illustrate the range of different speci fi c physiological endpoints that may apply, we present examples for fi ve diseases, without going in to method-ological details in the following paragraphs.

Gene therapy for hemophilia, for which several animal models exist, would entail continual blood collection at, say, monthly time points. Blood would be analyzed by functional tests to assess blood coagulation and by measuring expression of coagulation factors VIII or IX (for hemophilias A or B, respectively) by antigen ELISA. In addition, the blood could be analyzed by chromogenic coagulation factor activity assay. Finally, endpoint analysis could be performed to detect the coagulation factor in cells by immunohistochemistry, bearing in mind that secreted coagulation factor may result in back-ground in the vascular lumen unless tissue is perfused ( 6 ) .

Detection of the coagulation factors, in particularly factor IX, can also serve as a surrogate endpoint in preparatory studies requiring long-term repeated monitoring of a secreted protein (see Subheading 3.5 ).

For ornithine transcarbamylase de fi ciency, the sparse fur-abnormal skin and hair mouse model (spf-ash) acts as a useful model—it remains relatively well but lacks hair until approximately a month of age. The fi rst readout for successful gene expression in this model would be early (i.e. normal) acquisition of hair.


For longitudinal analysis measurement of urinary orotic acid is standard. For endpoint analysis histochemical (rather than immu-nohistochemical) analysis of enzyme expression is a standard assay ( 14 ) .

For cystic fi brosis, various mouse models exist, several of which exhibit fatal gut blockage around birth or weaning. Survival would therefore be one of the most striking consequences of successful gene expression in the gut. However, even in the absence of improved survival, electrophysiological properties of the airways and intestine would be useful phenotypic markers of expression of cystic fi brosis transmembrane conductance regulating protein (CFTR). There are also several antibodies directed against mouse and human CFTR, which are available from the CFTR folding consortium ( http://www.cftrfolding.org/CFFTReagents.htm ), which could be used in immunohistochemistry.

For neuronopathic Gaucher disease, the mouse model gener-ated by the Karlsson laboratory is one of extreme severity, with all mice succumbing to motor neuron degeneration and death before 20 days of age ( 15 ) . Again, survival and weight gain would be the most salient features of successful gene delivery. In addition, direct measurement of glucocerebrosidase, the enzyme de fi cient in this disease, is possible by the use of a fl uorometric enzyme activity assay. The enzyme can also be detected by western blot and immu-nohistochemistry. Enzyme expression could also be detected indi-rectly by assay for glucosylceramide. Alternatively, surrogate markers may also be examined. For example, microglial activation is a hallmark of the brain in fl ammation which precedes death, therefore, immunohistochemical detection of activated microglia is useful and can be performed using an anti-CD68 antibody ( 15 ) .

The majority of marker gene constructs for performance of the fol-lowing assays are readily available from commercial or academic sequence repositories such as Origene ( http://www.origene.com/ ) and the DF/HCC DNA Resource Core ( http://plasmid.med. harvard.edu/PLASMID/ ).

1. Homogenizing pestles (disposable). 2. Lysis buffer (from β -gal ELISA kit, Roche, Indianapolis, USA;

#11539426001). 3. Bicinchoninic acid (BCA) reagents A and B (Pierce, Rockford,

USA; #23223 and 23224, respectively). 4. Albumin protein standard 2.0 mg/ml (Pierce; #23209). 5. Microtiter plate. 6. Plate reader.

2. Materials

2.1. Total Protein Determination

http://www.cftrfolding.org/CFFTReagents.htm

http://www.origene.com/

http://plasmid.med.harvard.edu/PLASMID/

http://plasmid.med.harvard.edu/PLASMID/


1. Monoclonal anti-GFP primary antibody (Abcam, Cambridge, UK; #ab1218).

2. Biotin conjugated secondary antibody (Abcam; #ab6658). 3. Streptavidin–horse radish peroxidase (HRP) (Invitrogen,

Paisley, UK; #SNN2004). 4. 3,3 ¢ ,5,5 ¢ -Tetramethylbenzemidine (TMB) (Sigma, Gillingham,

UK; #T0440). 5. 2.5 M H 2 SO 4 . 6. Bicarbonate buffer: 0.8 g Na 2 CO 3 + 1.465 g NaHCO 3 in

500 ml. 7. Wash buffer: 200 μ l Tween-20 in 400 ml phosphate-buffered

saline (PBS). 8. Block solution: 4 g bovine serum albumin (BSA) in 400 ml

PBS. 9. Microtiter plate. 10. Plate reader.

1. Iso fl uorane. 2. Heparinized PBS. 3. Stereoscopic fl uorescence microscope. 4. Digital microscope camera. 5. Image analysis software.

1. Histoclear (National Diagnostics, Atlanta, GO, USA; #HS-200). 2. Industrial methylated spirits (IMS). 3. Deionized water. 4. 0.01 M citrate buffer, pH 6. 5. Tris-buffered saline (TBS). 6. 1% H 2 O 2 (Sigma; #H1009) made up in TBS. 7. Blocking solution: TBS containing 0.3% Triton X-100 (TBS-T)

and 15% normal goat serum (NGS) (Vector Laboratories, Burlingame, USA; #S-1000).

8. Rabbit polyclonal anti-GFP antibody (Abcam, Cambridge, UK; #ab290).

9. Biotinylated goat anti-rabbit IgG (Vector Laboratories; #BA-1000).

10. VECTASTAIN Elite ABC kit (Vector Laboratories; #PK-1000). 11. 0.05% 3,3-Diaminobenzidine (DAB) solution (Sigma;

#D8001) containing 0.01% H 2 O 2 . 12. DPX mounting medium (Sigma; #44581). 13. Microscope.

2.2. Green Fluorescent Protein

2.2.1. GFP ELISA

2.2.2. Direct Immuno fl uoresence Detection

2.2.3. Immuno-histochemical Detection


1. Iso fl uorane. 2. D -Luciferin (Goldbio, St. Louis, USA; #LUCK-1G). 3. Light-tight chamber: speci fi cally a Caliper Life Sciences IVIS

machine (see Note 1). 4. Cooled charge-coupled (CCCD) camera (as included in the

IVIS machine above). 5. LivingImage (Xenogen) software.

1. Lysis buffer (5×) (from Luciferase Assay System, Promega, Madison, USA; #E1500).

2. Luciferase assay reagent (from Luciferase Assay System). 3. Luminometer tubes. 4. Vortex. 5. Centrifuge. 6. Luminometer.

1. β -Gal standards. 2. β -Gal enzyme stock solution. 3. Anti- β -gal-digoxigenin (DIG). 4. Anti-DIG-peroxidase (POD). 5. POD substrate, 2,2 -azino-bis(3-ethylbenzthiazoline-6-

sulfonic acid) (ABTS). 6. ABTS substrate solution with enhancer. 7. Washing buffer: containing 10× PBS. 8. Sample buffer: containing PBS and blocking reagents. 9. Anti- β -gal-coated microtitre plate. 10. Plate cover. 11. Plate reader.

1. 70% Ethanol. 2. Stain 1: 0.169 g potassium ferrocyanide (Sigma; #P9387),

0.132 g potassium ferricyanide (Sigma; #P8131), 0.01 g MgCl 2 made up in 100 ml PBS—protect solution from light and store at room temperature.

3. Stain 2: 40 mg 5-bromo-4-chloro-3-indolyl- β - D -galactopy-ranoside (X-gal) (Goldbio, St. Louis, USA; #X4281C) in 1 ml DMSO.

4. PBS. 5. 10% Formalin. 6. Shaker.

2.3. Fire fl y Luciferase (Luc)

2.3.1. Whole-Body Bioimaging

2.3.2. Luminometry of Tissue Lysates

2.4. b -Galactosidase

2.4.1. b -Gal ELISA (from b -Gal ELISA Kit)

2.4.2. X-Gal Staining of Tissues


1. Capture antibody: polyclonal af fi nity puri fi ed anti-FIX antibody (Af fi nity Biologicals, Ancaster, USA; #EIA-FIX).

2. Detecting antibody: peroxidase-conjugated polyclonal anti-FIX antibody (Af fi nity Biologicals; #EIA-FIX).

3. Coating buffer: 1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 in 1 l distilled water, adjust buffer to pH 9.6, store for up to 1 month at 2–8°C.

4. PBS. 5. hFIX protein standard (Cambridge Biosciences, Cambridge,

UK; #HCIX-0040). 6. Wash buffer: 1 ml Tween-20 in 1 l PBS, adjust to pH 7.4. 7. Sample diluent (HBS–BSA–EDTA–T20): 5.95 g HEPES,

1.46 g NaCl, 0.93 g Na 2 EDTA, 2.5 g BSA, dissolved in 200 ml H 2 O, add 250 μ l Tween-20 and adjust solution to pH 7.2—make solution up to a fi nal volume of 250 ml with distilled water, aliquot, and store at −20°C.

8. Substrate buffer: 2.6 g citric acid and 6.9 g Na 2 HPO 4 —make solution up to a fi nal volume of 500 ml with distilled water, store for up to 1 month at 2–8°C.

9. o -Phenylenediamine 2HCl (OPD) substrate: 5 mg OPD in 12 ml substrate buffer, add 12 μ l 30% H 2 O 2 —make fresh for each assay.

10. Stopping solution: 2.5 M H 2 SO 4 . 11. Microtitre plate. 12. Plate reader.

1. Reagent 1: human factor X and FVIII:C (from Biophen Factor IX chromogenic assay kit, Hyphen Biomed, Neuville-sur-Oise, France; #221802).

2. Reagent 2: XIa-thrombin-calcium-phospholipids (from Biophen Factor IX chromogenic assay kit).

3. Reagent 3: chromogenic substrate SXa-11 (from Biophen Factor IX chromogenic assay kit).

4. Reagent 4: Tris–BSA buffer (from Biophen Factor IX chro-mogenic assay kit).

5. Abnormal plasma (internal control) (Hyphen Biomed; #223301).

6. Normal plasma (standards) (Hyphen Biomed; #223201). 7. Citric acid (2%). 8. Microtitre plate. 9. Plate reader.

2.5. Human Factor IX

2.5.1. hFIX Antigen ELISA

2.5.2. hFIX Chromogenic Activity Assay


1. SEAP assay buffer: 1.0 M diethanolamine pH 9.8, 0.5 mM MgCl 2 , 10 mM L -homoarginine.

2. Diluted p -nitrophenylphosphate in SEAP assay buffer (to a fi nal concentration of 120 mM).

3. Microcentrifuge. 4. Microtitre plate. 5. Plate reader.

1. Mammalian Genomic DNA Preparation Kit (Sigma–Aldrich). 2. Water bath or heating block. 3. Scalpel. 4. Ice-cold ethanol (95–100%). 5. Microcentrifuge.

1. Quantitative PCR machine. 2. PCR primers. 3. qPCR reagents: SYBR Green Mastermix (Roche), deoxyNTPs

(2.5 mM) (NEB).

It is often useful to quantify the total amount of protein present in tissue samples as the amount of transgene protein expression can then be expressed per mg of total protein. This allows the degree of transgene expression in different tissues to be compared directly.

1. Dissect the tissues and freeze in lysis buffer. 2. Defrost and homogenize the tissue with a homogenizing pestle

(see Note 3). 3. Spin for 2 min at 13,000 × g , keep the supernatant, and remove

cell debris. 4. Dilute the supernatant (depending on the concentration of the

sample) with fi nal volume >20 μ l. 5. Add 10 μ l of each protein standard (2, 1.5, 1.0, 0.75, 0.5,

0.25, 0.125, 0 mg/ml) and each sample to a microtitre plate (in duplicate).

6. Mix 50 parts BCA reagent A to 1 part BCA reagent B and add 200 μ l of this to each well.

7. Leave at room temperature for 10–15 min. 8. Read on a plate reader at 570 nm.

2.6. Secreted Alkaline Phosphatase

2.6.1. SEAP Activity Assay (See Note 2)

2.7. Vector Detection

2.7.1. Isolation of DNA from Mammalian Tissues

2.7.2. Vector Detection and Quantitation by PCR

3. Methods

3.1. Total Protein Determination


9. Use the BSA standard samples of known concentration to generate a standard curve.

10. Using the standard curve, determine the total protein concen-tration in the samples in mg/ml (see Notes 4 and 5 ).

GFP has become one of the more popular marker genes in recent years and there have been numerous modi fi cations to increase expression and to achieve fl uorescence at different wavelengths. It can be detected readily by ELISA, direct fl uorescence, and by immunohistochemical detection of tissue sections and is therefore very useful for endpoint analysis.

1. Dilute the primary antibody in bicarbonate buffer (1:10,000 monoclonal anti-GFP).

2. Add 100 μ l of the diluted primary antibody per well, cover the plate, and incubate at 4°C overnight.

3. Discard the antibody solution and wash 3× with 300 μ l wash buffer per well.

4. Add 300 μ l of block solution to each well, cover the plate, and incubate at 37°C for a minimum of 1 h.

5. Discard the block solution and wash 3× with 300 μ l wash buf-fer per well.

6. Add 100 μ l of standards to each well (in duplicate). To make up the standards, dilute standard GFP protein to a concentra-tion of approximately 4,000 pg/well. Do serial dilutions from here (300 μ l of dilution in 300 μ l wash buffer, etc.) down to 5–10 pg/well. Also include a buffer blank sample.

7. Dilute the protein samples so they contain between 2 and 200 μ g/well.

8. Add 100 μ l of protein sample per well (in duplicate). 9. Cover the plate and incubate at 37°C for 1 h. 10. Discard the standards and samples and wash 3× with 300 μ l

wash buffer per well. 11. Dilute biotin conjugated secondary antibody in block solution

(1:5,000 polyclonal anti-GFP). 12. Add 100 μ l of diluted secondary antibody to each well, cover

the plate, and incubate at 37°C for 1 h. 13. Discard the diluted secondary antibody and wash 3× with

300 μ l wash buffer per well. 14. Dilute the streptavidin–HRP in block solution (1:20,000). 15. Add 100 μ l diluted streptavidin–HRP to each well, cover the

plate, and incubate at 37°C for 1 h. 16. Discard the diluted streptavidin–HRP solution and wash 3×

with 300 μ l wash buffer per well.

3.2. Green Fluorescent Protein

3.2.1. GFP ELISA


17. Add 100 μ l of peroxidase substrate (TMB) to each well. Incubate for 10 min at room temperature. Avoid placing the plate in direct light.

18. Add 100 μ l of 2.5 M H 2 SO 4 to each well, in the same order as the other reagents were added, to stop the enzymatic reaction.

19. Read the plate using an ELISA plate reader at 450 nm within 30 min.

20. Use the GFP standard samples of known concentration to gen-erate a standard curve.

21. Using the standard curve, determine the amount of GFP pro-tein in the samples and express the value as pg GFP protein/mg of total protein (see Notes 4 and 5 ).

1. Anesthetize the mice using iso fl uorane. 2. Perform whole-body perfusion using heparinized PBS. 3. Dissect tissues of interest. 4. Place sample under stereoscopic fl uorescence microscope for

GFP visualization by means of a digital microscope camera. 5. Analyze the images using image analysis software.

1. Dewax paraf fi n embedded sections by immersing slide in Histoclear for 5 min. Repeat once.

2. Rinse with 100% industrial methylated spirit for 5 min. Repeat once.

3. Rinse the slides using deionized water. 4. Perform antigen retrieval by boiling sections for 10 min in

0.01 M citrate buffer. 5. Once the sections have cooled, rinse the slides in deionized

water and subsequently treat with 1% H 2 O 2 made up in TBS for 30 min.

6. Place sections in TBS-T/15% NGS blocking solution for 30 min.

7. Dilute rabbit polyclonal anti-GFP antibodies (1:1,000) in TBS-T/10%NGS.

8. Using a grease pen, draw a perimeter around the tissue and then add rabbit anti-GFP antibodies. Incubate overnight at 4°C.

9. Wash sections in TBS for 5 min. Repeat twice. 10. Dilute goat anti-rabbit IgG (1:1,000) in TBS-T/10% NGS. 11. Incubate sections in diluted goat anti-rabbit IgG for 2 h. 12. Dilute Vectastain avidin–biotin solution (1:200) in TBS. Make

this solution up to 30 min prior to use.

3.2.2. Direct Fluorescent Detection

3.2.3. Immuno-histochemical Detection


13. Rinse sections in TBS for 5 min. Repeat twice. 14. Incubate sections in diluted Vectastain avidin–biotin solution

for 2 h. 15. Rinse sections in TBS for 5 min. Repeat twice. 16. Apply 0.05% DAB solution containing 0.01% H 2 O 2 to the sec-

tions to visualize immunoreactivity. 17. Rinse the sections twice in ice-cold TBS in order to stop the

reaction. 18. Dehydrate the sections by submerging in rising concentrations

of industrial methylated spirits. 19. Clear the sections by immersing in Histoclear for 20 min.

Repeat once. 20. Add DPX mounting medium and overlay section with a cover-

slip. Allow to dry overnight in a fl ow hood. 21. Microscopically visualize DAB staining.

Luciferase describes a family of light-emitting compounds, how-ever, fi re fl y luciferase has proven to be the most popular as there is little background light emission and the substrate is relatively cheap. It is ideal for longitudinal monitoring of gene expression through whole-body bioimaging and it is also possible to perform endpoint analysis by luminometry of tissue lysates. Unlike with GFP however, immunohistochemical detection in tissue sections and analysis by ELISA are relatively poor.

1. Anesthetize the mice using iso fl uorane (see Note 6). 2. Administer D -luciferin through intranasal, intravenous, intrap-

eritoneal, or subcutaneous routes (see Note 7). 3. Place the anesthetized mouse in the light-tight chamber. 4. Take an image 5 min after administration of D -luciferin using a

charge-coupled device (CCD) or CCCD camera (see Note 8). 5. Perform data acquisition and analysis with LivingImage

(Xenogen) software.

1. The luciferase assay system should be stored at −20°C, and must be equilibrated at room temperature (20–25°C) for 30 min before use.

2. Perfuse the tissues with PBS to remove as much blood as pos-sible as hemoglobin may interfere with the assay.

3. Immerse approximately 1/8 of the liver in 200 μ l of 1× reporter lysis buffer.

4. Homogenize tissue using a pestle and mortar or an electronic homogenizer.

3.3. Fire fl y Luciferase

3.3.1. Whole-Body Bioimaging

3.3.2. Luminometry of Tissue Lysates


5. Centrifuge sample at 13,000 × g for 2 min at room temperature to pellet the cellular debris.

6. Transfer supernatant to clean eppendorf tube. 7. Using a nontransparent 96-well plate, pipette 20 μ l superna-

tant into each well. 8. Add 100 μ l luciferase assay reagent. 9. Place plate in the luminometer and initiate reading at the

appropriate wavelength depending on the type of luciferase being assayed.

β -Gal has been used extensively and so detection by both ELISA and by histochemical staining of whole tissues are well established. The motivation for choice of this marker gene would likely be the availability of preexisting constructs.

1. Determine the total protein content of the samples (see Subheading 3.1 ).

2. Ensure that all reagents are at 15–25°C before starting the test.

3. Dilute the protein samples in sample buffer to obtain 2–200 μ g protein per well and add 200 μ l to anti- β -gal-coated microtitre plate.

4. Dilute the β -gal standards (1.250, 0.625, 0.312, 0.156, 0.078, 0 ng/ml) and add to anti- β -gal-coated microtitre plate.

5. Cover the plate and incubate at 37°C for 1 h. 6. Discard the standards and samples and wash 3× with 300 μ l

wash buffer per well. 7. Dilute reconstituted anti- β -gal-DIG (50 μ g/ml) in sample

buffer to a fi nal concentration of 0.5 μ g anti- β -gal-DIG/ml sample buffer.

8. Add 200 μ l diluted anti- β -gal-DIG to each well, cover the plate, and incubate at 37°C for 1 h.

9. Discard the solution and wash 3× with 300 μ l wash buffer per well.

10. Dilute reconstituted anti-DIG-POD (20 U/ml) in sample buffer to a fi nal concentration of 150 mU anti- β -gal-DIG/ml sample buffer.

11. Add 200 μ l diluted anti-DIG-POD to each well, cover the plate, and incubate at 37°C for 1 h.


13. Add 200 μ l substrate with enhancer into each well and incu-bate at 15–25°C for 15–40 min (until a green color can be detected by eye).

3.4. b -Galactosidase

3.4.1. b -Gal ELISA


14. Read the plate using an ELISA plate reader at 405 nm. 15. Use the β -gal standards of known concentration to generate a

standard curve. 16. Using the standard curve, determine the amount of β -gal protein

in the samples and express the value as pg β -gal protein/mg of total protein (see Notes 4 and 5 ).

1. Dissect the tissues of interest and rinse thoroughly in PBS. 2. Immerse the tissue in 100% ethanol to fi x it for at least 1 h (see

Note 9). 3. Wash tissue 3× in PBS. 4. Mix stains 1 and 2 and add to samples making sure the tissues

are covered. 5. Incubate tissues on a shaking table at room temperature for

between 15 min and overnight. 6. Remove staining solution and wash cells 3× in PBS. 7. Place sample under stereoscopic microscope for visualization

of the blue precipitate (produced by the reaction of the X-gal substrate with the β -gal enzyme) by means of a digital micro-scope camera.

8. Analyze the images using image analysis software. 9. For permanent preservation, fi x the tissues in formalin.

hFIX is both a physiological endpoint when applying gene therapy to animal models of Hemophilia B or to hemophilic patients and a useful surrogate marker for longitudinal analysis of functional activity of gene therapy constructs. This is most relevant as a simple model for gene expression of a secretory protein using long-term minimally invasive monitoring by repeated bleeds. There are a vari-ety of commercial factor IX antibodies therefore ELISA can be readily performed.

1. Dilute the capture antibody (1/100) with coating buffer and add 100 μ l/well. Incubate at room temperature for 2 h.


3. Dilute the hFIX protein standard so the highest standard would be equivalent to ~100% of normal hFIX levels then dilute this 1/50 in sample diluent. Do serial dilutions from here (300 μ l of dilution in 300 μ l wash buffer, etc.) down to <1% hFIX. Also include a buffer blank sample.

4. Dilute the plasma samples 1/50 in sample diluent (see Note 10). 5. Add 100 μ l of standards and samples to wells (in duplicate)

and incubate at room temperature for 90 min.

3.4.2. X-Gal Staining of Tissues

3.5. Human Factor IX

3.5.1. hFIX Antigen ELISA



7. Dilute the detecting antibody (1/100) in sample diluent. 8. Add 100 μ l of diluted detecting antibody to each well and

incubate the plate at room temperature for 90 min. 9. Discard the solution and wash 3× with 300 μ l wash buffer

per well. 10. Add 100 μ l freshly prepared OPD substrate per well, allow

5–10 min for enzymatic reaction to occur and for color to develop.

11. Add 50 μ l 2.5 M H 2 SO 4 to each well to stop the enzymatic reaction.

12. Read the plate using an ELISA plate reader at 490 nm. 13. Use the hFIX standards of known concentration to generate a

standard curve. 14. Using the standard curve, determine the amount of hFIX pro-

tein in the samples and express the value as % of normal hFIX levels (see Note 4).

1. Reconstitute reagents R1, R2, and R3 with 2.5 ml distilled water per vial; reconstitute normal and abnormal plasma with 1 ml distilled water. Allow them to equilibrate at room tem-perature for 30 min, shaking occasionally.

2. Dilute the normal plasma 1/50 with Tris–BSA buffer (this is the highest standard and is equivalent to ~200% hFIX (see kit insert for precise concentration)). Perform serial dilutions to 1–5% hFIX. Also include a buffer blank sample.

3. Dilute samples (see Note 10) and abnormal plasma (see Note 11) 1/100 with Tris–BSA buffer.

4. Incubate samples, standards and reagents at 37°C for 15 min (see Note 12).

5. Add 50 μ l of standards and samples to the plate (in duplicate).

6. Add 50 μ l R1 to each well. Incubate at 37°C for 2 min. 7. Add 50 μ l R2 to each well. Incubate at 37°C for 3 min. 8. Add 50 μ l R3 to each well. Incubate at 37°C for 3 min. 9. Add 50 μ l citric acid to each well to stop the reaction. 10. Read the plate using an ELISA plate reader at 405 nm. 11. Use the hFIX standards of known concentration to generate a

standard curve. 12. Using the standard curve, determine the amount of hFIX

activity in the samples and express the value as % of normal hFIX activity (see Note 4).

3.5.2. hFIX Chromogenic Activity Assay


Human placental secreted alkaline phosphatase (hSEAP) is the most common form of this marker gene however alternative forms, such as murine SEAP, have shown promise, speci fi cally in mice.

1. Heat 250 μ l plasma at 65°C for 5 min (see Note 13). 2. Clarify medium by centrifuging at 14,000 × g for 2 min. 3. Add 100 μ l SEAP assay buffer and 100 μ l of clari fi ed medium

to each well of the microtiter plate and mix. 4. Warm the samples in the plate to 37°C for 10 min. 5. Pre-warm p-nitrophenylphosphate to 37°C. 6. Add 20 μ l pre-warmed 120 mM p -nitrophenylphosphate to

each well and incubate at 37°C. 7. Read plate in 1 min intervals at an absorbance of 405 nm using

the luminescence plate reader. 8. Use the change in absorbance over time to determine the lin-

ear reaction rate. 9. One milliunit of SEAP enzyme is de fi ned as the amount

of enzyme required to catalyze the formation of 1 nmol p -nitrophenol per hour.

Mammalian cells and tissues are lysed with a chaotropic salt-con-taining buffer to ensure denaturation of macromolecules. DNA is bound to the spin column membrane and the remaining lysate is removed by centrifugation. A fi ltration column is used to remove cell debris. After washing to remove contaminants, the DNA is eluted with buffer into a collection tube.

Before beginning the procedure, do the following: 1. Preheat a water bath or shaking water bath to 55°C. 2. Thoroughly mix reagents (any precipitate can be dissolved at

55–65°C). 3. Dilute wash solution concentrate with ethanol (95–100%). 4. Dissolve Proteinase K in water to obtain a 20-mg/ml stock

solution. DNA isolation using the GenElut Kit (Sigma–Aldrich)

1. Cut and weigh approximately 25 mg of tissue and mince with a scalpel.

2. Add lysis solution T followed by the Proteinase K solution to the tissue. Mix by vortexing. Incubate the sample at 55°C until the tissue is completely digested and no particles remain. Vortex occasionally or use a shaking water bath. Digestion is usually complete in 2–4 h. After digestion is complete, vortex brie fl y.

3. Add RNase A solution and incubate for 2 min at room temperature.

3.6. Secreted Alkaline Phosphatase

3.6.1. SEAP Activity Assay

3.7. Vector Detection

3.7.1. Isolation of DNA from Mammalian Tissues


4. Add lysis solution C to the sample and vortex thoroughly. Incubate at 70°C for 10 min.

5. Add column preparation solution to the GenElute Binding Column and centrifuge at 12,000 × g for 1 min. Discard fl ow-through liquid.

6. Add 200 ml of ethanol (95–100%) to the lysate; mix thoroughly by vortexing 5–10 s. A homogeneous solution is essential.

7. Transfer the contents of the tube into the treated binding col-umn from step 4. Centrifuge at 6,500 × g for 1 min. Discard the collection tube containing the fl ow through liquid and place the binding column in a new 2 ml collection tube.

8. Add 500 ml of wash solution to the binding column and cen-trifuge for 1 min at 6,500 × g . Discard the collection tube con-taining the fl ow-through liquid and place the binding column in a new 2 ml collection tube.

9. Repeat the wash and centrifuge for 3 min at maximum speed (12,000–16,000 × g ) to dry the binding column. The binding column must be free of ethanol before eluting the DNA. Centrifuge the column for one additional minute at maximum speed if residual ethanol is seen. Finally, discard the collection tube containing the fl ow through liquid and place the binding column in a new 2 ml collection tube.

10. Pipette the elution solution directly into the center of the bind-ing column, centrifuge for 1 min at 6,500 × g to elute the DNA.

Real time qPCR detects quantity of nucleotide sequence target by using speci fi c oligonucleotides homologous to the target sequence along with the incorporation of fl uorescent SYBR Green label to the double stranded product. SYBR Green (such as Power SYBR Green Master Mix—Applied Biosystems; P/N 4367659) is a dye that can bind to the minor groove of dsDNA, and become highly fl uorescent on excitation. In general terms, the reaction mix (SYBR Green Master Mix, forward primer, reverse primer and PCR grade water is prepared in a MicroAmp Optical 96-well Reaction Plate (AB; P/N N801-0560)) with each reaction prepared in triplicate. The plates are sealed with Optical Adhesive Covers (AB; P/N 4360954) and the qPCR reaction run on a real-time qPCR machine (such as a Sequence Detection 7500 system—Applied Biosystems) following the preprogrammed ampli fi cation parameters. Data out-put is expressed as an algorithm output (ddCt value) which indi-cates the ampli fi cation threshold for each reaction. These can either be expressed in comparison to a known and stable control or quanti fi ed in comparison to ampli fi cation of a series of standards.

SYBR green kits and methodologies vary with respect to the real-time PCR machine used (Applied Biosystems 7500, Roche LightCycler, Stratagene Mx3000), the fi nal quantitation process

3.7.2. Vector Detection and Quantitation by PCR


(ampli fi cation comparisons to a quanti fi ed standard curve or to a housekeeping gene such as GAPDH), and the primers used, there-fore, the authors advise to follow manufacturer’s instructions.

1. There are several whole-body bioluminescence imaging machines available from different companies, however, we pre-fer the IVIS machines from Caliper Life Sciences which have been extensively calibrated to permit comparison between machines.

2. There is both a chromagenic and a chemiluminescent assay available for SEAP; we have chosen the former as a standard spectrophotometer which is likely to be available in a wider range of labs. However, it is useful to bear in mind that the chemiluminescent assay is the most sensitive.

3. An electronic homogenizer can be used if the tissue samples size and lysis buffer volume are >500 μ l. Take care to clean the homogenizer carefully between samples.

4. All sample values must fall within the limits of the standard curve (if samples lie outside the standard curve limits, they must be diluted and the assay repeated).

5. Variable background is detected in some tissues; therefore, controls with un-transfected tissues of the type being analyzed should always be included.

6. We have observed that prolonged anesthesia can reduce the light emission and there have been some publications describ-ing this phenomenon ( 16 ) .

7. Although intraperitoneal injection has been the most widely used route of injection, there is evidence that other routes are more suitable. Particularly for general application, the subcuta-neous route is believed to result in more stable and prolonged light emission than other routes. In addition, we have observed that intranasal delivery is best for genes expressed within the lung ( 9 ) .

8. Ideally, the kinetics of light emission and the concentration of luciferin administered to the animal should be determined for each experiment; this is likely a cost–bene fi t analysis as higher concentrations of luciferin are likely to give greater light out-put but may be prohibitively expensive.

9. We observe no problems with fi xing overnight in ethanol at 4°C. Some protocols describe fi xation with formalin, para-formaldehyde, or gluteraldehyde but we did not fi nd them any better for tissue fi xation.

4. Notes


10. Plasma samples should be used fresh or immediately after defrosting but should not be freeze/thawed more than once.

11. Abnormal plasma is pooled human plasma at a known but less than physiological concentration (see kit insert for precise con-centration) so it can be used as an internal control for the assay if treated the same as a sample.

12. The plate should be placed in a water bath for the assay. 13. This is to eliminate background alkaline phosphatase activity.

References

1. Eglitis MA, Kantoff P, Gilboa E et al (1985) Gene expression in mice after high ef fi ciency retroviral-mediated gene transfer. Science 230:1395–1398

2. Morral N, O’Neal W, Zhou H et al (1997) Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: comparison of E2a wild type and E2a deleted vectors. Hum Gene Ther 8:1275–1286

3. Berger J, Hauber J, Hauber R et al (1988) Secreted placental alkaline phosphatase: a pow-erful new quantitative indicator of gene expres-sion in eukaryotic cells. Gene 66:1–10

4. Christou C, Parks RJ (2011) Rational design of murine secreted alkaline phosphatase for enhanced performance as a reporter gene in mouse gene therapy preclinical studies. Hum Gene Ther 22:499–506

5. Naylor LH (1999) Reporter gene technology: the future looks bright. Biochem Pharmacol 58:749–757

6. Waddington SN, Nivsarkar M, Mistry A et al (2004) Permanent phenotypic correction of Haemophilia B in immunocompetent mice by prenatal gene therapy. Blood 104:2714–2721

7. Misteli T, Spector DL (1997) Applications of the green fl uorescent protein in cell biology and biotechnology. Nat Biotechnol 15:961–964

8. Wu JC, Sundaresan G, Iyer M et al (2001) Noninvasive optical imaging of fi re fl y luciferase reporter gene expression in skeletal muscles of living mice. Mol Ther 4:297–306

9. Buckley SMK, Howe SJ, Rahim AA et al (2008) Luciferin detection after intra-nasal vector delivery is improved by intra-nasal rather than intra-peritoneal luciferin administration. Hum Gene Ther 19:1050–1056

10. Branchini BR, Ablamsky DM, Davis AL et al (2010) Red-emitting luciferases for biolumi-nescence reporter and imaging applications. Anal Biochem 396:290–297

11. Branchini BR, Ablamsky DM, Murtiashaw MH, et al (2006) Thermostable red and green light-producing fi re fl y luciferase mutants for biolumi-nescent reporter applications. Anal Biochem 22:499–506

12. Liu HS, Jan MS, Chou CK et al (1999) Is green fl uorescent protein toxic to the living cells? Biochem Biophys Res Commun 260:712–717

13. Bell P, Vandenberghe LH, Wu D et al (2007) A comparative analysis of novel fl uorescent proteins as reporters for gene transfer studies. J Histochem Cytochem 55:931–939

14. Mian A, McCormack WM Jr, Mane V et al (2004) Long-term correction of ornithine transcarbamylase de fi ciency by WPRE-mediated overexpression using a helper-dependent aden-ovirus. Mol Ther 10:492–499

15. Enquist IB, Bianco CL, Ooka A et al (2007) Murine models of acute neuronopathic Gaucher disease. Proc Natl Acad Sci U S A 104: 17483–17488

16. Franks NP, Jenkins A, Conti E et al (1998) Structural basis for the inhibition of fi re fl y luciferase by a general anesthetic. Biophys J 75: 2205–2211

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Prenatal Gene Therapy || Choice of Surrogate and Physiological Markers for Prenatal Gene Therapy