EP0946878A1 - Optimisation de fourniture de genes et de systemes de fourniture de genes - Google Patents

Optimisation de fourniture de genes et de systemes de fourniture de genes

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Publication number
EP0946878A1
EP0946878A1 EP97951337A EP97951337A EP0946878A1 EP 0946878 A1 EP0946878 A1 EP 0946878A1 EP 97951337 A EP97951337 A EP 97951337A EP 97951337 A EP97951337 A EP 97951337A EP 0946878 A1 EP0946878 A1 EP 0946878A1
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EP
European Patent Office
Prior art keywords
peptide
nucleic acid
approximately
immobilized
constant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97951337A
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German (de)
English (en)
Inventor
David Richard Thatcher
Paula Elizabeth Wilks
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Cobra Therapeutics Ltd
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Cobra Therapeutics Ltd
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Filing date
Publication date
Priority claimed from GBGB9626992.3A external-priority patent/GB9626992D0/en
Application filed by Cobra Therapeutics Ltd filed Critical Cobra Therapeutics Ltd
Publication of EP0946878A1 publication Critical patent/EP0946878A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • This invention is generally in the field of DNA delivery systems. More specifically, this invention describes methods of optimizing the efficiency of transfecting cells with a DNA molecule hound to;peptides and derivatives thereof.
  • Transfection of cells involves not only delivery of the- transfecting DNA to the cell nucleus, but also expression of the delivered DNA in the cell.
  • Some gene delivery systems involve transfection of cells using a delivery complex in which DNA is condensed with cationic polymers such as cationic lipids or polylysine (see, for example, Cotten and Wagner (1993) Curr. Opin. Biotech., 4: 705).
  • cationic polymers such as cationic lipids or polylysine
  • transfection techniques demonstrate that the transfecting DNA may be absorbed by cells, only to accumulate unchanged in the cytoplasm and unexpressed in the nucleus (for example, see Zaubner et al., (1995) J. Biol. Chem. 270; 18997, wherein cationic liposomes carrying DNA accumulate in the cytoplasm), suggesting that there is a need in the art for improvements in current methods of cell transfection.
  • This invention provides methods of screening of polyvalent cationic peptides, or derivatives of peptides (also called conjugates), for the ability to optimally transfect cells with a nucleic acid molecule condensed on the cationic peptide or conjugate based on determining the equilibrium dissociation constant (also called the equilibrium constant), K D . or the apparent dissociation constant (also called the apparent dissociation constant), for the interaction between the peptide or conjugate and the nucleic acid molecule.
  • the equilibrium dissociation constant also called the equilibrium constant
  • K D the apparent dissociation constant
  • the apparent dissociation constant also called the apparent dissociation constant
  • the invention is based on the recognition that the equilibrium constant for the interaction i between a peptide, 'or derivative thereof, and a nucleic acid molecule is readily determined by measuring the change in surface plasmon resonance (SPR) signal caused by a bimolecular interaction between the nucleic acid molecule which is immobilized on a metal sensor chip, and the peptide or conjugate which is present in a solution flowing over the chip, and that the equilibrium constant, or simply the apparent dissociation constant, is a critical predictor of optimal transfection efficiency.
  • SPR surface plasmon resonance
  • the terms “optimal transfection” or “optimally transfect” refers to obtaining the largest number of transfected cells per unit amount of nucleic acid condensed on a particular peptide or conjugate of a gene delivery complex. It will be appreciated by one of skill in the art that the proportion of transfected cells will vary for each target cell and therefore the largest number of transfected cells in a given target cell population may be, for example, 25% or 50% of the targeted cells for one selected cell population, and may be 80% of even as high as 95% for another selected cell population.
  • transfection is meaningful only in terms of expression of the nucleic acid in the host cell, and thus “expression” refers to the biological activity of the nucleic acid delivered to the host cell, whether that biological activity be the production of a gene product such as a protein or an RNA, or via a measurable biological effect of the nucleic acid in the host ceil.
  • the nucleic acid to be delivered to the host cell need not be identical in length or composition to the; nucleic acid immobilized on the sensor chip. It is preferred that the nucleic acid to be delivered and the chip immobilized nucleic acid possess the same overall properties as to DNA or RNA and as to strandedness (double or single-stranded).
  • the overall lengths of the delivered and immobilized nucleic acids be approximately the same; that is, if the nucleic acid to be delivered is about 50 nucleotides in length, the immobilized nucleic acid be in the range of 10 - 500 nucleotides in length; if the nucleic acid to be delivered is abou t 1.Okb in length, the immobilized nucleic acid be in the range of l.Okb - lOkb in length; if the nucleic acid to be delivered is about 50kb in length, the immobilized nucleic acid be in the range of I Okb - lOOkb in length.
  • the nucleic acid molecule immobilized on the sensor ship may be a DNA or an RNA, although DNA is preferred, and it may be a double-stranded or single-stranded nucleic acid molecule.
  • the immobilized nucleic acid may be of any desired length (e.g., generally in the range of 10 nucleotides - Ikb - 50kb), and may be of any sequence composition.
  • the nucleic acid molecule immobilized on the sensor chip is a double-stranded DNA which is a plasmid.
  • Plasmid refers to a circular or linear form of double-stranded DNA which is large enough to contain a gene (that is, a sequence comprising coding as well as regulatory regions).
  • the invention thus encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprising detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to a solution of a test peptide, wherein the change in surface plasmon resonance occurs upon binding of the peptide to and dissociation of the peptide from the immobilized nucleic acid, thereby to permit either calculation of the equilibrium dissociation constant K ⁇ or apparent dissociation constant, k din; and (b) selecting the peptide having an equilibrium dissociation constant, K D , with a value of approximately 1 x 10" 12 to 1 x 10' s (or apparent dissociation constant, * with a value of approximately 1 x 10"* to 1 x 10 * ')-
  • the invention also encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprising (a) detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to (a) a solution containing a test peptide for a time sufficient to permit binding of the peptide to the immobilized nucleic acid followed by (b) a solution lacking test peptide for a time sufficient to permit dissociation of the peptide from the immobilized nucleic acid, wherein the change in surface plasmon resonance occurs upon binding of the peptide to and dissociation of the peptide from the immobilized nucleic acid, thereby to permit calculation of the equilibrium dissociation constant K D ; and selecting a peptide having an equilibrium dissociation constant, K 3 , with a value of approximately 1 x 10' 12 to 1 x 10 * *.
  • the invention also encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprising (i) detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to (a) a solution containing a test peptide for a time sufficient to permit binding of the peptide to the immobilized nucleic acid followed by (b) a solution lacking test peptide for a time sufficient to permit dissociation of the peptide from the immobilized nucleic acid, wherein the detection detects a change in surface plasmon resonance which occurs upon dissociation of the peptide from the immobilized nucleic acid, thereby to permit calculation of the apparent dissociation i constant, k ⁇ ; and (ii) selecting a peptide havmg an apparent dissociation constant, kj, with a value i of approximately lj x 10 " * to i x 10°.
  • the methods further comprise, prior to the detecting step, the steps of immobilizing the nucleic acid on a sensor chip; and exposing the immobilized nucleic acid molecule to a solution of a test peptide; and, optionally, the step of recording the change in surface plasmon resonance.
  • the invention also encompasses a method of screening test peptides for the ability to optimally transfect cells with a nucleic acid molecule comprisng (a) immobilizing the nucleic acid on a sensor chip; (b) exposing the immobilized nucleic acid molecule to a solution of a test peptide; (c) detecting and recording a change in the surface plasmon resonance upon binding of the peptide to the immobilized nucleic acid; (d) calculating the equilibrium dissociation constant K D (or apparent dissociation constant, kj) of the test peptide from the recorded change in surface plasmon resonance; and (e) selecting the peptide having an equilibrium dissociation constant, K , with a value of approximately 1 x 10" 12 to 1 x 10 "6 (or apparent dissociation constant, * with a value of approximately 1 x 10-* to 1 x 10 *1 ).
  • the invention also encompasses a method of transfecting cells with nucleic acid comprising the step of providing a nucleic acid condensing pcptide/condensed nucleic acid complex, wherein a peptide of the complex has an equilibrium dissociation constant, K D , with a value of approximately 1 x 10" 12 to 1 x 1 "*, or an apparent dissociation constant, k ⁇ , with a value
  • the invention also encompasses a method of transfecting cells with nucleic acid comprising delivering to the cell a nucleic acid condensing peptide/nucleic acid complex wherein a peptide of the complex has an apparent dissociation constant, k d , with a value of approximately 1 x IO to 3 x 10' 3 or an apparent dissociation constant, k d , is approximately I x 10- 2 to 3 x l0-'.
  • the invention also encompasses a method of measuring the equilibrium constant, K D (or apparent dissociation constant, k for a peptide binding to plasmid nucleic acid comprising (a) immobilizing the plasmid nucleic acid on a sensor chip; (b) exposing the immobilized nucleic acid molecule to a solution of a test peptide; (c) detecting and recording a change in the surface plasmon resonance:upon binding of the peptide to the immobilized nucleic acid; and (d) calculating the equilibrium constant K D (or apparent dissociation constant, k ⁇ ) of the test peptide from the recorded dhange in surface plasmon resonance.
  • the invention also encompasses improv ents upon a method of delivering a recombinant nucleic acid to a population of host cells, the improvement comprising obtaining optimal transfection of the cells by determining the equilibrium constant K D of a peptide that is present in a complex comprising a nucleic acid condensing peptide and a condensed nucleic acid for transfection from a change in surface plasmon resonance which occurs upon binding of the peptide to and dissociation of the peptide from an immobilized nucleic acid, or wherein the improvement comprises obtaining optimal transfection of the cells by determining the apparent dissociation constant, I ⁇ , of a peptide that is present in a complex comprising a nucleic acid condensing peptide 1 and a condensed nucleic acid for transfection from a change in surface plasmon resonance which occurs upon dissociation of the peptide from an immobilized nucleic acid.
  • the methods may further comprise selecting an equilibrium rate constant, K D , with a value of approximately 1 x 10' 12 to 1 x 10'* or apparent dissociation constan , k d , with :a value of approximately 1 x 10 *6 to 3 x 10 *3 .
  • the screening methods of this invention comprise the additional step of selecting peptides or conjugates for transfection having an apparent dissociation constant, k ⁇ , in the range of approximately 1 x 10 4 to 1 x 10" 2 sec -1 or an equilibrium constant, K D , in the range of approximately 1 x 10" 9 to 1 x 10" 2 mol" 1 .
  • the screening methods of this invention comprise the additional step of selecting peptides or non-lipid conjugates for transfection having an apparent di ⁇ flooi ⁇ tion oonfltant, k discipline, in the range of approximately 1 x 10" to 1 x 10' 2 see 1 or an equilibrium constant, Kp, in the range of approximately 1 x 10"* to 1 x 10' 1 mol -1 for use in vivo transfections where it is important that decondensation of the nucleic acid from the peptide or conjugate does not occur as the nucleic acid-peptide or nucleic acid-conjugate complex is diluted, such as when injected into the blood stream.
  • the highest transfection efficiency is using lipopeptides having K D *s in the range of 1 x 10' s to 1 x 10" 7 (or s of l x lO-' to l x lO "2 ).
  • the screening methods of this invention comprise selecting peptides or conjugates for transfection having an apparent dissociation constant, k d , of approximately 1 x 10" 2 sec *1 or an equilibrium constant, K D , of approximately 1 x 10' 6 mol" 1 for use in in vivo transfections where the nucleic acid-peptide or nucleic acid-conjugate complexes are administered directly to a localized area of tissue, such as a tumor.
  • This invention also provides a method of determining the apparent dissociation constant, l j, or the equilibrium dissociation constant, K D , for a peptide or conjugate binding to or complexing with plasmid nucleic acid comprising immobilizing the plasmid nucleic acid on a metal sensor chip, exposing the immobilized plasmid nucleic acid molecule to a solution containing a peptide or conjugate, detecting and recording a change in the surface plasmon resonance of the peptide or conjugate-nucleic acid interaction, and calculating the apparent i dissociation constant, kj, or the equilibrium dissociation constant, K D .
  • the invention also encompasses a composition of matter comprising a sensogram (i.e., data which is rendered in tangible form such as on a piece of paper) in which the Y axis of the seniOgram corresponds to resonance unite and the X axis corresponds to time and the data line corresponds to a recording over time of a detected change in surface plasmon resonance of a bimolecular interaction between a nucleic acid molecule and a peptide or conjugate.
  • a sensogram i.e., data which is rendered in tangible form such as on a piece of paper
  • the Y axis of the seniOgram corresponds to resonance unite
  • the X axis corresponds to time
  • the data line corresponds to a recording over time of a detected change in surface plasmon resonance of a bimolecular interaction between a nucleic acid molecule and a peptide or conjugate.
  • the invention also encompasses a sensogram in which the Y axis of the sensogram corresponds to resonance units and the X axis corresponds to time and the data line corresponds to detecting a change in the surface plasmon resonance of a nucleic acid immobilized on a sensor chip and exposed to a solution of a test peptide, wherein the change in surface plasmon resonance occurs upon binding of the peptide to and dissociation of the peptide from the immobilized nucleic acid.
  • This invention permits selection of peptides having sufficient binding capacity for nucleic acid to provide a gene delivery complex that is stable in the bloodstream, targetable to selected tissue types, and capable of efficient transport into the cytoplasm and to the nucleus. These requirements are met by selecting peptides based on their equilibrium constants as taught herein.
  • the invention thus! permits one of skill in the art to provide a condensing peptide/condensed nucleic acid complex wherein a proportion of the nucleic acid condensing peptides have linked thereto a selected functional group such as a cell targeting ligand, a cell import sequence, or a nuclear import sequence.
  • Fig. 1 is a diagram of a theoretical sensorgram of resonance units (RU) versus time of a bimolecular interaction between a nucleic acid molecule and a peptide or conjugate, the three characterisitic regions of the sensogram, and a maihetical equation describing each of the three regions.
  • RU resonance units
  • Fig.2 is a sensogram for the interaction of the peptide NBC11 with plasmid nucleic acid pRSVluc.
  • Fig.3 is a sensogram for the interaction of the Lip9 conjugate with plasmid nucleic acid pRSVluc.
  • Fig.4 is a graph of equilibrium dissociation constant, Q , versus the number of positive charges per mole of peptide for plasmid pRSVLuc nucleic acid.
  • Fig. 5 is a graph of equilibrium constant versus lueiferfase activity of cells transfected with plasmid pRSVLuc nucleic acid complexed with NBC9 series peptides.
  • FIGs. 6 A and 6B show results of immunostaining of tumor cells using nucleic acid condensing peptide/nucleic acid complexes according to the invention.
  • Figure 6A shows the results of transfection of Mouse KLN205 Tumour with plasmid pTXO 118 complexed with 0.5 ⁇ g NBC15 peptide/ ⁇ g DNA.
  • Figure 6B shows the results of transfection of Mouse KLN205 Tumour with plasmid pTXO 118 complexed with 0.5 ⁇ g NBC9 peptide/ ⁇ g DNA.
  • Fig. 7 shows the relative transfection efficiency of individual NBC peptides and polylysine peptides.
  • Fig. 8 is a graph showing the effect of the presence of lipidated nucleic acid condensing peptide on the transfection efficiency of a gene delivery system as described herein.
  • Fig. 9 shows the transfection efficiency of complexes wherein the nature of the lipidated peptide was varied in complexes made up of 0.6 ⁇ g lipidated peptide 2 ⁇ g free.
  • This invention provides methods of screening peptides for the ability to optimally transfect cells with a gene or other nucleic acid molecule.
  • Such peptides can be used as the peptide component of an efficient non-viral gene delivery system useful in in vitro, ex vivo, or in vivo methods of gene delivery, such as in gene therapy.
  • Control of decondensation is therefore an important parameter to consider in the design of gene delivery systems in order to achieve optimal levels of successful transfection, that is levels of transfection provide for clinically or diagnostically useful levels of transfection in a target cell population.
  • "Useful" levels of transfection are determined by the clinician or diagnostian for a given disease or disease parameter, and thus are determined by one of skill in the art for a given nucleic acid, a given target ceil, and a given disease.
  • a nucleic acid condensing peptide/nucleic acid delivery complex may comprise peptides having different amino acid compositions, and 'therefore different affinities for the nucleic acid and/or different functional groups attached to the peptide, and therefore different affinities for the nucleic acid.
  • a given complex may comprise a nucleic acid condensed with a first peptide having a hydrophilic coatings to promote evasion of the reticular endothelial system, a second peptide targeting end ⁇ some escape functions, a third peptide having a nuclear import sequence, and a fourth peptide having no functional group attached.
  • the first and second peptides are useful for entering the cell and cell cytoplasm, but are not required once the complex has entered the cytoplasm.
  • the third peptide is useful for entering the nucleus, but is not required after nuclear entry.
  • the fourth peptide is useful throughout the cell targeting and entry process, and ultimately must be released upon decondensation of the nucleic acid in the cell nucleus.
  • the four peptides will have the same arnino acid sequence, but will differ in affinities for the nucleic acid by virtue of the functional group attached to the peptide.
  • the four peptides may have different amino acid sequences, thereby providing for additional variations in affinities for the nucleic acid to be delivered.
  • a critical factor in determining whether a first peptide-nucleic acid complex will transfect cells better than a second complex is the ability of the peptide component of the complex to release from the condensed nucleic acid component, i.e., thereby to promote decondensation, at an appropriate cellular location (either the cytoplasm or the nucieoplasm) for the particular nucleic acid to be expressed.
  • an accurate measure of the affinity of the peptide component for the nucleic acid is required.
  • conjugate refers to a derivative of a peptide which contains additional functional groups that are covalently linked or conjugated to a core peptide.
  • Such functional groups may enhance the nucleic acid condensing or decondensing ability of the core peptide or enhance, the transport and delivery of the nucleic acid-peptide complex to the appropriate cellular location for subsequent decondensation and transcription of the nucleic acid component of the complex.
  • Examples of such functional groups include lipids (see below).
  • peptide component encompasses the use of conjugates in a gene delivery system and that the term “peptide-nucleic acid complex” includes conjugate-nucleic acid complexes as well.
  • SPR Surface plasmon resonance
  • reproducible data can also be obtained on the binding of cationic peptides to immobilized nucleic acid.
  • conditions required for complete dissociation of a peptide from the nucleic acid can be determined.
  • different peptides are ranked in order of their avidity for nucleic acid as dete ⁇ nined by the i equilibrium dissociation constant, K & , or the apparent dissociation constant, kj, calculated from the SPR measurements using a biosensor-based device, such as the BIAcore. Such a ranking is directly correlated .with the ability of each peptide to efficiently transfect cells with the nucleic acid.
  • SPR was detected using the BIAcore system.
  • This machine contained an SPR detector and an integrated microfluidic cartridge which is engineered such that an analyte solution (cationic peptide) is continuously passed over the sensor chip.
  • the sensor chip consists of a glass plate coated with a thin film of gold covered in a layer of carboyxymethyl dextran.
  • the chip used has a dextran layer to which streptavidin is coyalently attached.
  • the chip is prepared for use by adding biotinylated nucleic acid which will be bound to the chip via the strfeptavidin-biotin interaction, and the same chip is used in a series of experiments.
  • the SPR As material binds to the immobilized nucleic acid molecule on the sensor chip, the SPR
  • Changes in angle tire defined as resonance units (RU) and data are collected every second. Background effects are detected and automatically subtracted by the use of a control flow cell in line with the nucleic acid flow cell.
  • the data is displayed as a sensorgram which plots the change in signal with time.
  • a typical sensorgram has three phases: an association phase, an equilibrium phase and a dissociation phase (see Fig. 1).
  • the rise in signal occurs as more peptide binds to the immobilized plasmid DNA until equilibrium conditions are reached.
  • the height of the peak is directly proportional to the mass of peptide bound. If the flow continues with buffer containing no peptide, peptidd begins to dissociate from the complex and the resonance signal is reduced.
  • the sensorgram can be used to compute apparent rate constants for each of the three individual processes.
  • P s is the amount of bound peptide, k, is the apparent rate constant for association
  • Equation 2 shows that the equilibrium rate constant, K Q , can also be calculated from these data.
  • N C2 Is a peprlde containing Three sequence motifs -based on Histone H1 , NBC15, NBC8, NBC9 and NBC10 contain 1, 2, 3 and 4 sequence repeats dariv ⁇ d from Hieton ⁇ HI .
  • NBC7 and NBC11 are, respootivoiy, arginino rich and lyainc rich peptides designed to form -helical structures which will interact with the major groove of the DNA helix.
  • the pseudo-first order rate constants for the association reaction i.e., the "on rate”
  • the association reaction is more complex with clear biphasic kinetics (Fig.2).
  • a fraction of the peptide associates with nucleic acid with a higher binding rate and a fraction with a lower rate.
  • Dissociation rates decrease with increasing molecular size.
  • K D is a measure of the relative affinity of the peptide for nucleic acid under equilibrium conditions. In general, for a related series of cationic peptides, D decreases proportionally with increasing size.
  • the sequence of the nucleic acid condensing peptide and whether it has one or more attached functional: groups has a marked effect on transfection efficiency. Without being bound to any one theory, it is believed that transfection efficiency is partly dependent on the relative affinity of the nucleic acid condensing peptides for nucleic acid. It is believed that, unless nucleic acid binding affinity is relatively high, the complex will decondense too soon upon entry into the cell, e.g., in the cytoplasm of the cell, and be unavailable for nuclear import in the ceil.
  • Nucleic acid condensing peptides useful in the invention possess the following characteristics.
  • Nucleic acid condensing peptides useful in the invention are characterized in that the peptides are heteropeptides.
  • heteropeptide refers to a peptide having a selected amino acid sequence.
  • the term “heteropeptide” refers to a peptide having an amino acid composition of at least two different amino acids. This is in contrast to a "homopeptide” in which the amino acid composition consists of identical amino acids.
  • a homopeptide consists of 100% of the same amino acid
  • a heteropeptide consists of a peptide chain in which as few as a single amino acid differs from the remainder of the amino acids in the chain, or, for example, 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90%, etc., of the amino acids in a chain differ from the remaining amino acids in the chain.
  • a heteropeptide is therefore a linear peptide consisting of, in terms of its selected amino acid sequence, a variety of (i.e., at least two, at least three, at least four, etc.,) types of amino acids.
  • amino acid refers to any of the twenty natural commonamino acids, and also may refer !to natural, uncommon amino acids or amino acid derivatives or analogs.
  • An amino acid in D- or L- form may be present in a peptide according to the invention.
  • Nuqleic acid condensing peptides of the invention are also characterized in that a plurality of nucleic acid condensing peptides or a preparation of nucleic acid condensing peptides useful in the invention has a low polydispersion index.
  • Nucleic acid condensing peptide preparations of the prior art e.g., known as polycations, have a polydispersion index >1.1. This is evident in the description of polylysine peptide preparations in theiprior art as having a "mean Mr" (Wu et al., (1987) J. Biol. Chem., 262: 4429; Wu et al., (1988) J. Biol. Chem., 263: 14621; and Wu et al., (1988) Biochem., 27: 887) or having "an average chain length" (Wagner et al., (1991) Proc. Nat. Acad.
  • Such preparations are disclosed as having an average degree of polymerization of a given number of lysine groups, e.g., "polylysine 90” has an average degree of polymerization of 90 lysine groups; “polylysine 190” has an average degree of polymerization of 190 lysine groups; etc (Bimsteil et al., supra.)
  • Preparations of polylysine are available commercially from Sigma Corporation (St. Louis, Missouri) and are known by one of skill in the art and documented upon purchase to have an actual distribution of sizes within each sample whichj varies per sample, and may vary, for example, from 30% - 150% of the material being distributed.
  • the actual peptide length within such a sample may be, for example, 80% above or 80% below the stated average length of the peptide.
  • the length of the peptide is thus reported as an average of various sizes which average is determined by low-angle light scattering analysis of individual lots of chemically synthesized peptide.
  • Nucleic acid condensing peptide preparations useful according to the invention have a low polydispersion index (PDI), also termed index of polydtspersity.
  • PDI polydispersion index
  • a "low" polydispersion index refers to a PDI of ⁇ 1.01.
  • Nucleic acid condensing peptides useful in the invention have a PDI ⁇ 1.01 , and preferably have a PDI ⁇ 1.001.
  • Nucleic acid condensing peptide preparations of the invention may have a PDI of 1.0 and thus are termed "monodispersed”.
  • a "high" polydispersion index refers to a PDI of 1.1, >1.2, >1.3, and generally in the range of 1.1 - 2.0.
  • the polydispersion index is used to characterize the molecular weight distribution of polymeric compounds.
  • the PDI for a polymer having a homogeneous distribution of molecular weights i.e., where the polymer preparation contains a uniform molecular weight
  • the value approaches 2.0.
  • the polydispersion index of a peptide preparation may be determined according to two methods which are described below: I) using light scattering and colligative properties of the peptide preparation to determine the weight and number average molecular weights of the peptides in a preparation; or 2) using electro-spray mass spectrometry to determine the molecular weights of peptides in a given preparation. It is critical to the invention that the PDI be calculated according to the most accurate of the two methods, i.e., electro-spray mass spectrometry.
  • the former method of calculating the PDI provides only a rough estimate of the PDI in that, for a given peptide preparation, the ratio of weight average molecular weight / number average molecular weight for heterogeneous polymers may be determined, respectively, by light scattering and from colligative properties of the peptide preparation (Q. Odian in Principles n olymerisation. John Wiley and Sons, (1981)).
  • colligative property is viscosity (Odian, supra).
  • the PDI for polymer peptides disclosed herein may be calculated from analysis of the peptides by electroi-spray mas3 spectrometry. This method gives the exact mass of each component to within 0.001%.
  • the PDI values of the peptide preparations useful in the present invention are in therange of 1.0 - 1.01. Peptide preparations which are especially useful in the invention possess a PDI ⁇ 1.01 , and even ⁇ 1.001. In contrast, the PDI values for the nucleic acid condensing peptide preparations reported in the prior art are >1.1.
  • the polydispersion index is used to characterize the molecular weight distribution of polymeric compounds.
  • Mw is the weight average molecular weight and Mn is the number average molecular weight.
  • the usual method for determining the weight average molecular weight of heterogeneous polymers such as poly-lysine is to use light scattering where the amount of light scattered depends on molecular size as well as the number of complexes.
  • the number average molecular weight is calculated from a colligative property of the polymer in solution such as viscosity since this property is dependent on the number of molecules of polymer per unit volume.
  • Poly-L-Lysine 25000; Sigma Product P7890, Lot No 93H-5516 has a PDI of 1.2. This result is typical for the batches of poly-lysine used in many gene transfer experiments reported in the literature. For preparations of low polydispersity this approach cannot be used because of the I relative inaccuracy of both viscosity and light scattering measurements. In these cases electrospray mass spectrometry may be used.
  • the deconvoluted electrospray mass spectrum of peptide NBC9 (theoretical mass 4082.2) was determined as follows. An aqueous solution of lmg/ ml peptide was prepared and the sample diluted in a mixture of acetonitrile:methoxyethanol:0.1% trifluoroacetic acid. The experiment was performed on a VG Instruments Quattro II instrument fitted with a quadropole analyser. The instrument was calibrated with m ⁇ globin and lO ⁇ l aliquots were injected direc ⁇ y into the instrument source.
  • a nucleic acid condensing peptide suitable according to the invention is a basic peptide, i.e., a peptide with net positive charge, for example a peptide or polypeptide comprising 2 - 50, preferably 12 - 40, and more preferably 15 - 38 D- or L- amino acid residues.
  • the polypeptide includes at least 30%, more preferably 50%, and could include as much as 80 - 90% basic amino acids, such as lysine, arginine, histidine, ornithine or a non-natural amino acid containing a side chain having a secondary or tertiary amine group.
  • a nucleic acid condensing peptide according to the invention is preferably a peptide of, for example, 8 - 50 residues in length which includes a cysteine and/or threonine and or serine residue which is available for regio-specific conjugation to a ligand, as defined herein. It is preferred that the cysteine and threonine residues be located N* and C-terminally, respectively, such that they may! act as handles for covalent attachment of ligands.
  • nucleic acid condensing peptides useful in the invention also include peptides wherein a j large proportion (eig., 30% - 90%) of the amino acid composition of the peptide is a single basic amino acid species, such as lysine.
  • a heteropeptide which includes a sequence of from approximately 8 to approximately 30 lysyl residues is useful according to the invention.
  • a peptide which is a homopolymer of lysyl residues is not useful according to the invention because homopolymerio polylysine tends to be cytotoxic and to stick ncnspecif ⁇ cally to cell surfaces under certain conditions.
  • the heteropolymeric nucleic acid condensing peptides contemplated herein are advantageous over homopolymers such as polylysine.
  • a preferred nucleic acid condensing peptide is a peptide or polypeptide having an alpha-helical ( ⁇ -helical) conformation structure.
  • Such peptides have been designed to interact with nucleic acid by interacting through a conformational structure that is alpha helical.
  • a functional group is covalen ⁇ y linked to the peptide, it follows that the functional group of the conjugate may be positioned around the turn of the alpha helical structure so as to be optimally exposed to the outer surface of the nucleic acid-conjugate complex.
  • nucleic acid-peptide complexes i.e., nucleic acid noncovalently associated with a peptide or a conjugate which includes a peptide of the invention covalently linked to a functional group.
  • the relevant j intermolecular interactions are refined by building a rigid scaffold which will specifically dock with nucleic acid and allow positioning of the functional moiety such that steric hindrance will not occur.
  • a rigid scaffold is provided by any peptide which forms a stable ⁇ -helix.
  • a number of proteins that specifically interact with nucleic acid interact through an ⁇ -helix structure (10 residue) which lays across the major groove of the nucleic acid helix. This ⁇ -helix is usually followed in the polypeptide by a reverse turn forming the helix-turn-helix motif which is characteristic of these .proteins.
  • Most of the interactions thajt these sequences make with the DNA are weak hydrogen bonds to the phosphate and bases. This is because the function of these domains is to recognize a particular base sequence and then trigger a conformational change. We hypothesized that if such helices are designed so that the hydrogen bonds were replaced by ionic interactions, the binding would be much stronger and useful as a docking/condensation peptide.
  • a peptide having an ⁇ -helical scaffold is designed as follows. Using a molecular model of a 10 residue ⁇ -helix, a helical wheel and a model of B-DNA, it is apparent that strong interactions can be made using , where X, is a basic residue interacting with the phosphate on one side of the major groove and X 2 is a basic residue interacting with a phosphate diametrically opposite the other side of the major groove. Either of X 5 and X 2 may be, for example, lysine, arginine, or histidine.
  • Lysine, arginine, and histidine are preferred because both amino acids 1) are of sufficient size to bridge the major groove gap, 2) will interact strongly with phosphate and 3) are strong helix formers. If the residues BB are also residues with strong helix-forming propensities then the peptide will form a stable ⁇ -helix in solution.
  • nucleic acid condensing peptides according to this aspect of the invention will fall within the generic formula
  • X isia naturally occurring or synthetic amino acid carrying a positively charged group on the side chain such as lysine, arginine, 2,4-diamino-butyric acid, histidine, and omithine or a non->natural amino acid containing a side chain having a secondary or tertiary amine group;
  • V is naturally occurring amino acid which has a high propensity to promote alpha helix formation as defined by Wilt and Thornton ((1988) J. Mol.
  • Biol., 203: 2221-232 such as glutamic acid, alanine, lcucine, methionine or glutamine, tryptophan or histidine; where Z are naturally occurring amino acids with at least 3 members of the sequence having a high propensity to form stabilized turn structures as defined by Wilt Thornton (he.
  • peptides useful in the invention may contain one or more internal serine, threonine, or cysteine residues, preferably at a position in the sequence which will be exposed for conjugation to a selected ligand, and thus not on the positively charged (DNA oriented) face of the ⁇ -helix. This positioning of selected reactive amino acid residues within the peptide and oriented such that they do not contact the face of the peptide that contacts DNA permits conjugation of the peptide with other functional peptides by bonds of selected and defined stability.
  • Cysteine allows specific conjugation via the thiol side chain to compounds coffiaining other reactive thiol groups (via disulfides), alkylating functions (to form thioether bonds), or other thiol reactive groups such as malcimide derivatives.
  • Bonds of "defined stability" are described hereinbelow, and include bonds such as acid labile bonds (hydrazone) or linkages that are less stable in the reducing environment of the cytosol (disulfide). Such bonds are useful for carrying functional groups on the complexes.
  • a peptide will contain: 1) helix-forming amino acids, 2) a repeating three-dimensional structure that contacts the major groove of the DNA, 3) suitable chromophores for (ruantitation, and 4) a number of "handles" (i.e., reactive sites) for regio- specific conjugation of ligands which form accessory functional domains.
  • suitable chromophores for (ruantitation, and 4) a number of "handles” (i.e., reactive sites) for regio- specific conjugation of ligands which form accessory functional domains.
  • handleles i.e., reactive sites
  • the lysine at position 9 of NBC7 and the glutamic acid at position 9 of NBCl 1 are available for regie-specific conjugation via reductive amination, peptide bonds, etc., and the cysteine at position 16 for conjugation via thioether and disulfide bonds.
  • the C-terminal glutamic acid of NBC7 is available for modification to a hydrazide and therefore foi coupling via an oxime or hydrazone bond.
  • Ancjther preferred nucleic acid condensing peptide is a peptide comprising a sequence which is derived from a sequence of histone HI protein and other human histones.
  • Histone Hl' is a highly basic protein of the histone family that is found in all higher organisms. Histone HI unlike the majority of other members of this family does not assemble as an integral part of the nucleosome. Histone HI is believed to interact primarily with those stretches of chromosomal DNA linking the nucleosome complexes of chromatin (Allen et al., (1 80) Nature, 288 . Histone HI binds to naked DNA with the same salt dependence as HI depleted chromatin (Kumar and Walker, (2980) Nucleic Acids Research, 8: 3135).
  • Histone HI protein is not useful as a component of a gene therapy delivery vehicle i because it is not readily available and is a biological repressor of its own transcription. Recombinant histone HI cannot readily be produced by recombinant methods, and the protein is too large to be synthesized chemically. Piirification of HI from mammalian sources other than human cells poses a safety hazard.
  • histone HI to promote the condensation of plasmid DNA for transfection is likely to reduce expression of the delivered gene because HI is part of a general repressor mechanism where the presence of excess histone HI leads to reduced transcription (Weintraub, H., (1985) Cell, 705 - 711; Croston et al., (1991) Science 251: 643).
  • Nucleic acid condensing peptides of the invention may include those portions of HI (sequence I, II or III, below) which are identified herein as sequences which possess the ability to condense nucleic acid. Therefore, a nucleic acid condensing peptide of the invention can comprise a linear combination of the following three consensus sequences where the total sequence length is>17 residues: Sequence I:
  • K is Lysin ⁇
  • P is Proline
  • A is Alanine
  • X is Serine, Threonine or Proline
  • Y is Alanine, Proline, or Valine
  • Z is Alanine, Threonine, Lysine, or Proline
  • B is Lysine, Alanine, Threonine or Valine
  • J is Alanine or Valine.
  • X is Alanine or Valine
  • K is Lysine
  • S is Serine
  • P is Proline
  • A is Alanine.
  • X is Lysine or Arginine
  • Y is Alanine, Valine, or Threonine
  • Z is Proline, Alanine or Serine
  • B is Alanine, Lysine, Threonine or Valine
  • K is Lysine
  • P is Proline
  • A is Alanine.
  • Sequence IV is a consensus sequence from all human histone sequences:
  • A is preferably Lysine or Threonine: B is preferably Glycine or Glutamine; C is preferably Glycine, but can also be Aspartate, Glutamate, or Serine; D is preferably Glycine, but can also be Lysine, Valine, Glutamine, or Threonine; E is preferably Lysine or Alanine; F is preferably Alanine or Lysine, G is preferably Arginine, but can also be Valine or Isoleucine; H is preferably Alanine, but can also be Threonine, Histidine, or Proline; I is preferably Lysine, Arginine, or Glutamine; J is Alanine or Anginine; and K is preferably Lysine or Glutamine.
  • a preferred consensus sequence is:
  • NBCl has the following structure: NH 2 -[SV40 NLS]-[Seq I]-[Seq U]-[Seq ⁇ I]-[SV40 NLS>[Seq I]-C-COOH (SEQ IDNO:6).
  • -C- is Cyste ne;where the SV40 NLS has the sequence Pro-Lys-Lys-Lys-Arg-Lys-Val- Glu- (Dingwall and Laskey, (1991) Trends Biochem. Sci., 16: 478).
  • nucleic acid condensing peptide of the invention will have an amino acid sequence that falls within the following generic sequence:
  • X is either absent or Serine or Threonine
  • Y is sequence I (SEQ ID NO: 8), II (SEQ ID NO:9), III (SEQ ID NO:10) or IV (SEQ ID NO:17) as defined above
  • n is 2-6; and C is Cysteine.
  • Particularly preferred peptides are the following:
  • NBC2 has the structure: NH 2 -[Seq III]-[SV40 NLSl]-[Seq I]-C-COOH (SEQ ID NO: 11), where -C- is Cysteine.
  • NBC8 has the structure: NH [Con Seq I]-[Con Seq Ij-C-COOH (SEQ ID NO: 12) where -C- is Cysteine.
  • NBC13 has the structure: NII r [Seq IJ-[Seq I]-[Seq Ij-C-COOH (SEQ ID NO:13) where -C- is Cysteine.
  • NBC10 has the structure: NH r [Seq I]-[Seq I]-[Seq I]-[Seq H-C-COOH (SEQ ID NO:14) where -C- is Cysteine.
  • Sequences NBC8 - 10 are derived from part of NBC2, but lack the nuclear localization sequence and have a repeat motif (seq. I, II or III above) which has enabled us to look at the effect of peptide length on function.
  • NBC8 has a double repeat of this sequence, NBC 13 a triple repeat and in NBC10 the sequence is quadrupled.
  • NBC conjugates e.g., lipidated or insulin-derivatized NBCs are also described herein and according to chemical syntheses known to one of skill in the art.
  • Peptides NBCl - 2 and 4 - 14 were synthesised using a Biosearch 9050 plus Pepsynthesizer in extended synthesis cycle mode using Fmoc-Cys(Acm)-O «PEG-PS-Resm with dimethylformamide as solvent. Deprotection of the N-terminal Fmoc-group before each coupling was achieved using a solution of 20% piperidine in dimethylformamide (Imin at a high flow rate followed by lOmi s at 3 Vmin).
  • the arnino acid3 were coupled in four fold excess using O-(lH-benzotriazo-l-yl)*tetramethyluronium tetrafiuoroborate (TBTUyi-hydroxybenzotriazole and N-ethyidiisopropylamine as activating agents.
  • O-(lH-benzotriazo-l-yl)*tetramethyluronium tetrafiuoroborate (TBTUyi-hydroxybenzotriazole and N-ethyidiisopropylamine as activating agents.
  • the following amino acid derivatives were used as appropriate for the NBC: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc*Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ty ⁇ (tBu)-OH, Fmoc-Val-OH.
  • the N-terminal Fmoc group was removed as described above to give the free amino, side chain protected, peptide bound to the resin. Each resin was then washed into a glass vial with methanol and dried in vacua.
  • Peptides were cleaved from the resin using a TFA/waier/phenol/thioanisole/ 1,2-ethanedithiol (82.5: 5: 5: 2.5) mixture (10ml of mixture for every gram of resin to be cleaved). The resin was then removed by filtration and washed 3 times with TFA. The combined filtrate and washings were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to give the crude peptide.
  • NBCl was then desalted by dissolving it in 0.75ml of 1% acetic acid in water and applying it to a PD-10 column which was then eluted using the same buffer. Fractions were taken every 0.75ml and fractions 3 - 8 were combined and lyophilised to give the final peptide.
  • NBC2 and A - 14 were purified by preparative rp hplc, desalted by elution on a Sephadex G25 superfi e column (1.6x30cm) with 1% acetic acid in water. The resulting fractions were analysed by reverse phase hplc, pooled and lyophilised as above.
  • the acetamidomethyl (Acrn) thiol protecting group maybe removed using mercury (I ⁇ )iacetate in 30% acetic acid in water followed by precipitation of the mercury with 2-mercaptoethanol.
  • the resulting free thiol peptide can be purified using gel filtration to give the desired product.
  • a conjugate may include a peptide which has a lipid group attached to it, and thus forms a lipid-derivatized bucleic acid condensing peptide.
  • lipid is meant to refer to a four - thirty carbon mola ' cule that is insoluble in water and soluble in alcohol.
  • the term includes fats, fatty oils, essential oils, waxes, sterols, cholesterols, phospholipins, glycolipins, sulfolipins, aminolipins, chromolipins, and fatty acids.
  • the nucleic acid condensing peptide can be specifically modified according to the invention by condensation with an lipid, for example, an activated ester of a fatty acid.
  • the fatty acid is ideally either palmitic acid, oleic acid, such as dioleoylphospiiatidylethanol ⁇ mine, myristic acid, or cholesterol, although other fatty acids, such as stcaric acid, may also be employed.
  • Synthesis of NBC peptides conjugated to lipids are described in detail in the Examples. Th* effort of the preo ⁇ noe of the lipid component on tra ⁇ efiction ⁇ frioi ⁇ noy of the eompiox ia dramatic, leading to >40-£bld increase in activity.
  • the phenomenon is unrelated to the effect observed during DNA transfection using cationic lipids, where activity is proportional to the level of cationic lipid in the complex; in which the ligand density required for is orders of magnitude higher (e.g., lOx-lOOx) than the ligand density required for gene transfer using complexes provided according to the present invention.
  • lipidated peptides in the co ⁇ plex or the complex formulation are resistance of the complex to degradation.
  • the presence of lipidated peptide in the complex formulation confers higher resistance to inactivation by human plasma and increases the relative level of transfection after exposure of the complex to various levels of human plasma in the transfection medium.
  • Insulin was chemically modified as described herein and according to previously described methods! (pp.43 - 44 of Of ⁇ brd, R.E. (1980) "Semisynthetic Proteins", Wiley, Chichester and New York), with slight modifications. Briefly, 1 OOmg Zn-free insulin were dissolved in 1ml of 1M sodium hydrogen carbonate, diluted with 4mL dimethylformamide and reacted with an equixnolar amount (relative to peptide amino groups) of methy ulphonyloxycarbonio acid N-hydroxysuccinimide ester.
  • the reaction medium was acidified and diluted with 0,1 %TFA, and the derivatized insulin isolated by semi-preparative HPLC on a C8 column equilibrated in 0.1% TFA, using a 35 - 45% gradient (same eluants zs described above) over 20mins.
  • the methylsulphonyloxycarbonyl groups were then cleaved under standard conditions and the material repurified on the C8 column using a 35 - 40% gradient over 20mins.
  • the final compound, Boc-AoA-insulin was characterized by electrospray mass spectrometry (calcd. m/z 5950.6; found m z 5948.1 ⁇ 0.1) and was deprotected by TFA treatment (30 mins at room temperature) just before conjugation to a peptide, as described below.
  • Cell targeting components such as monoclonal antibodies may be conjugated to a nucleic acid condensing peptide via the carbohydrate group or via a hydrazone and disulfide i linkage or via a hydrazone and thioether linkage, or conjugation may provide a clustered antibody complex, all as described in detail below, to provide a complex for use in transfection according to the invention.
  • Glycosyl moieties may be clustered by the use of branched carbohydrates of natural or synthetic origin. Alternatively these groups may be clustered by modifying a branched amino acid backbone (dandrim ⁇ r).
  • glycosylated dendrimeric moieties described herein are based on a generic design of a ligand that, when conjugated to an NBC peptide, will give optimal binding of the peptide to either the hepatocyte asialoglycoprotein receptor (ASGPR) or the macrophage mannose receptor (MMR).
  • ASGPR hepatocyte asialoglycoprotein receptor
  • MMR macrophage mannose receptor
  • the type of monosaccharide used in the synthesis of the ligand will depend on the receptor of interest.
  • the design of the ligand is based on a consideration of the binding requirements of the ASGPR and the MMR, as it is these receptors that arc of primary interest for use in gene targeting.
  • Ligands which present several (i.e three or more) monosaccharide units clustered together with their non-reducing ends exposed generally provide optimal binding to membrane l ⁇ otinB, including the ASGPR and MMR (Lee and Lee, (1987) Glycoconjugate J., 4: 317 - 328; Monsigny et al., (1'994) Adv. Drug Del. Rev., 14: 1 - 24).
  • Such ligands bind with more affinity than corresponding ligands presenting only one or two monosaccharide units - the so-called "cluster effect".
  • the nucleic acid is made up to 20 - 400 ⁇ g/ml in the buffer.
  • Peptide or conjgate ⁇ g/ ⁇ g + lipidated peptide ⁇ g/ ⁇ g ( ⁇ g ⁇ g referring to ⁇ g peptide per ⁇ g DNA); DNA concentration; NaCl concentration.
  • Buffer is normally 25mM phosphate pH 7.4, but can be 25mM Hepes pH 7.4. The amount of complex required for the experiment is calculated in terms of volume and total mass of DNA. Th'e volumes of the different components required are calculated.
  • NBC peptide or conjugate at lOmg/ml or lmg ml; Lipidated peptide at lOmg/ml or lmg ml; Phosphate buffer at 0.5M; NaCl at ' 5M; DNA - concentration is variable, normally 1 - lOmg ml.
  • the total volume is made up with water.
  • Complexes of the invention will possess an overall (net) charge as follows. Complexes which are designed so as to target a particular cell type, and therefore contain a targeting ligand, will possess an overall charge in the range of 0.5 - 3.0, more particularly in the range of 0.5 - 2.0, and optimally in the range of 0.8 - 1.2. Particles which do not target a particular cell type, but are designed so as to transfect a broad range of cell types will possess an overall charge in the range of 0.5 - 5.0, more particularly in the range of 1.0 - 3.0, and optimally in the range of 1.3 - 3.0. The overall charge (i.e., the balance of positive and negative charge species) of the complex are determined as follows.
  • nM Positively Charged Groups ( ⁇ g Peptide Molecular Weight x 10-3) x No. of Positive Charges in Sequence.
  • Example 7 The nucleic acid condensing activity of peptides having different charges and the transfection efficiencies of complexes having different charge ratios are presented in Example 4. Trfln ⁇ . ⁇ *.rinn e Ytclencies of particles containin R varvinp; amounts of ratios of positively/negatively charged residues are presented in Example 7. Highly efficient transfection may be obtained using particles of the invention which do not contain a targeting iigand and are therefore untargeted with respect to a specific cell type. Such particles are highly efficient with respect to transfection where the ratio of positive negative charges is greater than 1.25.
  • the size of a virus like particle fall within the range of 5nm to 500nm. It has been found that the efficiency of uptake of the particle by a cell dramatically decreases when the particle size is greater than 500nm. This is likely due to the size of the endo ⁇ omal pores in a given cell type. Particle size is measure by lasar light scattering or at ⁇ miv f ⁇ rvc micr ⁇ ac ⁇ py, or electron microscopy. Therefore, the size of a particle of the invention will vary depending upon the cell type and the size of endosomal pores in a given cell type.
  • Ratio of nucleic acid condensing peptides/nucleic acid molecules A complex 1 according to the invention will have a ratio of the number of peptide the number of nucleic acid molecules in a particle that is within the range of 10/1 to 1,000,000/1. This ratio will depend upon the relative sizes of the peptide and nucleic acid molecules, the degree of condensing activity of the peptide, and the degree of condensation that the nucleic acid attains. More particularly, the range will be 100/1 - 10,000/1. For example, for NBC2 in combination with an 8kb vector, a useful ratio for untargeted delivery of the vector to cells is approximately 5000:1 (relative numbers of molecules).
  • a useful ratio for targeted delivery of the vector to cells is approximately 1000: 1.
  • the nucleic acid is an oligonucleotide of, e.g., 10 - 50 nucleotides in length
  • the ratio i of p ⁇ ptide/oligonucleotide is in the range of 0.1 - 10.0 and is preferably 0.5 - 1.0.
  • exogenous nucleic acid was transferred to mammalian cells in vivo.
  • a representative in vivo ceil transfection system is transfection of tumor cells using a murine carcinoma model.
  • the murine carcinoma model DB A2 containing the squamous epithelial syngenic tumour cell line KLK-205 was prepared as described in the Examples.
  • a transfecting complex was prepared using a solution of plasmid DNA encoding a reporter gene or, for example, nitroreductase and.a selected NBC peptide 0.5 ⁇ g peptide/ ⁇ g DNA, as described herein. Where desired, a conjugate peptide was added to the transfecting complex, at a concentration of 0, 1 - 0.3 ⁇ g peptide/ ⁇ g DNA.
  • Transfection efficiency may be determined in a number of ways, including assessing the effect of the reporter gene on the target tissue, or by quantitating the gene product. Below, tissues samples were obtained and nitroreductase gene expression was ascertained via staining of tissue sections. Determining Ex Vivo Transfection Efficiency via Systemic Administration of Transfected Cells.
  • cells are transfected ex vivo and reinjected into the animal. Reporter gene expression is then determined.
  • In vitro transfection efficiency is determined using plasmid DNA containing a marker gene for firefly luciferase (pRSVLuc; de Wet J.R., Wood K.V., DeLuca M., Helsinki D.R., and Subramani S., (1987) Mol. Cell. Biol., 7: 725 - 737). After incubation the cells are lysed and are assayed for gene expression. In the case of the luciferase reporter gene, luciferin and ATP are added to lysed cells and the light emitted is measured with a luminometer (see deWet et al., supra).
  • Cell lines such as Jurkat and K562 grow in suspension and can be transfected and the level of transgene expression (luciferase activity) determined by the following method.
  • Cells are harvested on the day of assay by centrifugation at 1200 rpm for 5mins at room temperature. The cell pellet is resuspended in phosphate buffered saline and re- centri uged This operation is performed twice. The cell pellet is then suspended in RPMI 1640 (Gibco Ltd.) to make up a suspension of 2.7 x 10* cells per ml. The cells are then aliquoted into tubes and 0.75 m!
  • RPMI medium containing 10% foetal bovine serum.
  • Each 0.5ml of transfected cell suspension is transferred to a well of a 12 well plastic culture plate containing 1 ,5ml of RPMI 10% FBS.
  • the original transfection tube is rinsed with a further 1 l of medium and the wash transferred to the culture dish making a final volume of 3ml.
  • the culture plate is then incubated at 37 °C for 24 - 90h in an atmosphere of 5% CO2.
  • the contents of each well in the culture dish are transferred to centrifuge tubes and the cells collected by centrifugation at 13 OOOrpm.
  • the pellet is resuspended in 0.12ml of Lysis Buffer (IOOmM sodium phosphate.
  • the lysaie is centrifuged at 13000 rpm for Imin and the supernatant collected. 80 ⁇ l of the supernatant are transferred to a luminometer tube. The luciferase activity is then assayed using a Berthold Lumat L9501 luminometer.
  • the assay buffer used is Lysis Buffer containing 1 OmM Luciferin and IOOmM ATP. Light produced by the luciferase is integrated over 4s and is described as relative light units (RLU). The data are converted to RLU / nil of lysate, RLU/cell or RLU/mg protein (protein concentration of the lysate having been determined in this case by the BioRAD Lowry assay.
  • Tne method can be modified for adherent cells such as the hepatic carcinoma cell line HepG2.
  • Cells were plated 24h prior to transfection in 6-well tissue culture plates at a density of 1 - 2 x 10 J per well in complete medium (MEM (with Earle's salts) ⁇ 1 % non- essential amino acids ⁇ 10% foetal calf serum).
  • Transfection was carried out by aspirating the culture medium and washing The cells with phosphate buffered saline. Complex (50 ⁇ g/m!
  • DNA is layered onto the cell monolayer in a 1ml final volume containing 2.5 ⁇ g DNA and 120 ⁇ M chloroquine in RPMI supplemented with human albumin (lmg/ml) and human transferrin (50 ⁇ g/ml).
  • the transfection is then allowed to proceed by incubating the cells at 37°C for 4h, after which the transfection complex was removed, the cells washed with PBS and completed medium added prior to incubation at 37 ⁇ C for 24 - 72h in an atmosphere of 5% CO2.
  • the cells were then harvested using trypsin to detach the cells. After addition of medium containing serum to inactivate the trypsin, cells were pelleted by centrifugation at 13,000 rpm. The cells were then suspended in 1ml of PBS and re-centrifuged.
  • the pellet was resuspended in 200 ⁇ l of Lysis Buffer (1 OOmM sodium phosphate, pH 7.8; 8mM MgC12, ImM EDTA; 1% Triton X-l 00; 15% glycerol; 0.5mM PMSF and ImM uitniotnreitoi and agitated witn a pipette, ine lysate is centntuged at u, ⁇ uu rpm for Imin and the supernatant collected. 80 ⁇ l of the supernatant are transferred to a luminometer tube.. The luciferase activity is then assayed using a Berthold Lumat L9501 luminometer.
  • the assay buffer used is Lysis buffer containing 0.0 ImM Luciferm and 0.04375mM ATP. Light produced by the luciferase is integrated over 4sec and is described as relative light units (RLU). The data are converted to RLU/ml of lysate, RLU/cell or RLU/mg protein (protein concentration of the lysate having been determined in this case by the BioRAD Lowry assay.
  • the relative transfection efficiencies observed in vivo can be correlated with the dissociation kinetics of peptide-nucleic acid bimolecular interactions measured using surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the conjugate When it is necessary to maintain high affinity for plasmid DNA (for example for transport of a gene into the nucleus using a conjugate peptide ⁇ ntaining nuclear localization functional group), the conjugate should have a k d approaching (i.e., less than or equal to) approximately 1 x 10 -4 sec -1 , or a K D of less than 1 x l0 -9 mol -1 .
  • Such peptides as NBC 10 (k d of approximately 1 x 10 *J sec -1 , K D of approximately 5.0 x 10" ⁇ mol" 1 ) and NBCl 1 (k d of approximately 1.6 x 10 "4 sec '1 , 0 of approximately 7.8 x 10"' mol'' or 5 x 10' 10 ) have such binding characteristics but are not completely deleterious to transfection because dissociation is able to occur efficiently within the cell, This is not the case with polylysine (PolyK) which has an extremely highiaffinity for nucleic acid (k ⁇ of approximately 1 x 10" 6 and K D of approximately 5 x 10" 12 and extremely poor transfection efficiency.
  • PolyK polylysine
  • the complex will comprise peptides or conjugates which which are substantially peptides or conjugate peptides having a k a in the range of approximately 1 x 10 " " 3 to 1 x 10" 4 sec' or a K D in the range of approximately 1 x 10 "8 to 1 x 10"'° mol" 1 .
  • complexes which comprise peptides or conjugates which are substantially peptides or conjugates having a kd of approximately 1 x 10 " * sec *1 or Kp of approximately 1 x 10* mol -1 , such as in peptides NBCl 5 and BC8.
  • the transfection efficiency of a given complex in vivo or in vitro can be predicted from the KD and also from the number of cationic groups in the peptide from the correlation shown in Fig. 5.
  • nucleic acid condensing peptides used i according to the invention (Example 1), the conjugation of functional groups to a condensing peptide, for example the conjugation of insulin to a nucleic acid condensing peptide, the conjugation of a lipid to a nucleic acid condensing peptide, the conjugation of a monoclonal antibody to a nucleic acid condensing peptide, and the conjugation of sugar groups to a nucleic acid condensing peptide (Examples 2 * 5); the determination of the affinity constant of a bimolecular interaction consisting of a nucleic acid condensing peptide and an immobilized nucleic acid using urface plasmon resonance according to the invention (Example 6); the assembly and formation of a nucleic acid condensing peptide/nucleic acid complex according to the invention (Example 7); and the testing of complexes for in vitro,
  • Peptides NBC1-2 and 4-14 were synthesised using a Biosearch 9050 plus Pepsynthesizer in extended synthesis cycle mode using Fmoc-Cys(Acm)-O-PEG-PS «Resin with dimethylformamide as solvent. Deprotection of the N-terminal Fmoc-group before each coupling was achieved using a solution of 20% piperidine in dimethylfcrmamide(l in at a high flow rate followed by 1 O in at 3ml/min). The amino acids were coupled in four fold excess usin
  • the resin was then 'removed by filtration and washed 3 times with TFA.
  • the combined filtrate and washings were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to gi,ve the crude peptide.
  • NBCl was then desalted by dissolving it in 0.75ml of 1% acetic acid in water and applying it to a PD*10 coiumn which was then eluted using the same buffer. Fractions were taken every 0.75ml and fractions 3 * 8 were combined and lyophilised to give the final peptide.
  • NBC2 and 4-14 were purified by preparative rp hplc, desalted by elution on a Sephadex G25 superfine column (1.6x30cm) with 1% acetic acid in water. The resulting fractions were analysed by reverse phase hplc, pooled and lyophilised as above.
  • the acetumidomethyl (Acm) thiol protecting group maybe removed using mercury (II) acetate in 30% acetic acid in water followed by precipitation of the mercury with 2-raercaptoethanol.
  • the resulting free thiol peptide can be purified using gel filtration to give the desired product.
  • InsIa B -NBC1 ' (superscripts refer to linkage site on ligand and peptide) has the following structure' :
  • NBC14 was conjugated to functionaiized insulin as follows.
  • the Cys-protected peptide was oxidized as follows. The peptide was dissolved in 50mM imidazole (CI), pH 6.9 at a concentration of 10 mg/mL, and 0.2 methionine in water was added (as an anti-oxidant scavenger), a 10-fold molar excess over peptide.
  • CI imidazole
  • pH 6.9 pH 6.9
  • methionine methionine
  • the isolated oxidized peptide was dissolved into a solution of 5mg of the AoA-insulin derivative (an approx 2-fold molar excess of peptide over insulin) made up in 0.5mL 0.1M sodium acetate buffer to which had been added 50 mL acetonttrile, followed by adjustment to pH 3.8 (glass elctrode) with glacial acetic acid.
  • the conjugate was isolated after 15h incubation at room temperature and characterized by electrospray mass spectrometry (calculated, m z 8426.1, found m/z 8429.3 ⁇ 0.5). 4rag of material were isolated by semi-preparative HPLC with a 30 - 45 % gradient from the bulk of the reaction mixture. 1 The peak fraction was dried down in a SpeedVac vacuum centrifuge. The final yield was 4mg of donjugate.
  • N- ⁇ , A' SC, N-e,B J0 - SC-insulin (referred to as MSC) 2 -Insulin) already used in the construction of AoA-Insulin is also an intermediate in the synthesis of insulin*PEG 3400 -AoA.
  • the material was then reacted with a 2-fold excess Boc-AoA-OSu to introduce an axninooxyacetyl group at the N-terminus of the PEG chain.
  • Conditions of reaction and purification were the same as in the previous step.
  • the isolated product (25mg) was then deprotected by successive treatment with NaOH and TFA, as already described for the synthesis of AoA-Insulin and repurified by semi-preparative HPLC on a C8 column with a 30 - 50%B2 gradient over 40mins.
  • This derivative could be characterized by matrix-assisted laser desorption ionisation MS on a Voyager-Elite time of flight instrument (PerSeptive Biosyste s).
  • An insulin derivative which lacks the B 2 ⁇ " 31 sequence (amino acids 23-31 from the amino terminus, "des-insulin") was prepared as follows: 70mg of insulin were dissolved in 7.0ml of 50mM Hepes buffer, pH 8.0 and digested with 7mg of TPCK-trypsin for 3h at 37. ⁇ C. The reaction was stopped by the addition of 140ml of a 0.1M solution of p-methyl sulphonyl fluoride in ethanol and the addition of acetic acid until a pH 3.0 was attained.
  • the des-insulin was isolated by preparative rp-hplc using a Waters Nova-Pak HR C18 column and an aqueous trifluoroacetic a ⁇ i ⁇ (0 ⁇ %) to acetomtrile trifluoroacetic acid/water (900:1:100) gradient (20-40% gradient over 40min). 38mg of material were recovered and characterized by electrospray mass spectrometry (calculated mass 4865.5 / observed mass 4867.7).
  • Periodate oxidized NBC 14 was prepared as described herein for NBC peptides. 4mg of this material were mixed with 4mg of des-insulin derivative in 400ml of O M sodium acetate buffer containing 20% acetonitrile, pH 3.6. After 15h incubation at room temperature the conjugate was isolated by preparative hplc using a 1cm diameter C8 column and a 30-35 % gradient of acetonitrile solvent (using Solvent A and Solvent B as described above). The recovered material (3.2mg) was characterized by electrospray mass spectrometry j (calculated mass 7465 / observed mass 7462).
  • N-Paimitoyl derivatives of the corresponding NBC peptides are called Lipl, Lip2, etc.
  • Lipl N-Paimitoyl derivatives of the corresponding NBC peptides
  • Lip2 N-Paimitoyl derivatives of the corresponding NBC peptides
  • N-palmitoyl-NBC2 (Lip 2) is as follows. The synthesis of any lipidated peptide may be adapted accordingly.
  • Palmitic acid N-hycfroxysuccinimide ester (0.5g per gram of resin) was added to a suspension of N-terminal amino deprotected resin bound NBC2 peptide (side chain protected)-0-PEG-PS resin in methanol chloroform (1:4; 5ml per gram of resin). The reaction was shaken for 48h at room temperature then the resin was filtered off, washed 3 times with chloroform, washed with methanol and dried in vacuo.
  • the peptide was cleaved from the resin using a TFA/water/ ⁇ henol/thioanisoie/1,2- ethanedithiol (82.5: 5: 5: 5: 2.5) mixture (10ml of mixture for every gram of resin to be cleaved).
  • the resin was then removed by filtration and washed 3 times with anhydrous trifluoroacetic acid.
  • the combined filtrate and wasltings were concentrated by evaporation then precipitated using diethyl ether followed by centrifugation to give the crude peptide.
  • the crude peptide was dissolved in a small amount of 25mM HEPES pH 7.4 and applied to an SP-Sepharose fast flow column of an appropriate size and eluted using a gradient of 0 - 2M NaCl in 25mM HEPES pH 7.4 over 20 column volumes.
  • the fractions containing the peptide, as shown by analytical reverse phase hplc, were pooled and applied directly to a preparative reverse phase hplc (Dynamax 83-221-C column) and eluted using an appropriate gradient of acetonitrile (0.1% TFA) in water (0.1% TFA) (typically 30 • 50% acetonitrile over 20min).
  • the fractions containing the desired peptide were pooled, the acetonitrile evaporated in vacuo and lyophilised.
  • the LIP-peptides were desalted by elution on a Sephadex G25 superfine column (1.6x30cm) with 1% acetic acid in water.
  • the resulting fractions were analysed by reverse phase hplc and those containing pure peptide were pooled and lyophilised to give the final peptide.
  • the Carbohydrate groups present on the monoclonal antibody are oxidized using periodic acid to produce reactive aldehyde groups (Jentoft and Dearborn, (1979) J. Biol. Chem., 254: 4359).
  • the oxidized antibody is then reacted with amino groups present on the NBC2 peptide. This results in the formation of an imine (Schiff Base) linkage between the antibody and the NBC polymer.
  • the imine linkage is very labile in water and must be reduced using sodium cyanoborohydride or sodium borohydride to give the stable amine.
  • Anti-CD7 antibody (50mg at 5mg/ml) in 25mM sodium acetate, pH 5.0 was oxidized by the addition of sodium periodate to lOmM. The solution was left on ice, in the dark for lh. The oxidized antibody was desalted and the buffer exchanged to 25mM MES, pH 6.0 on a Sephadex G25 column (30cm x 2.5cm i.d.) . The amount of antibody recovered from the column was determined by measuring the absorbance at 280nm (the absorbance of a lmg/ml solution of antibody at 280nm is 1.33). A 5 times molar excess of NBC 1 was added and the solution was left at room temperature for lh.
  • the unmodified antibody does not bind to the column and is wa ⁇ hed through.
  • the antibody conjugated to NBCl binds to the column and is eluted early in the gradient.
  • the conjugate peak eluted at approximately 1M NaCl.
  • the protein 1 fractions were pooled and stored at 4"C NBC-2-SH (5mg/ml) was reduced with dithiothreitol (dithiothreitol) and purified by gel filtration using 25mM HEPES, 0.5M NaCl, pH 7.9, as running, buffer. A total of 250nmol NBC2 (as determined using an Ellman's test) were added to the antibody-PDPH and the solution was left for 16h at room temperature. The concentration of NaCl was adjusted tn 0 1 W hfifnre the antibndy-NBC2 conjugate was purified by cation exchange on SP Sepharose using NaCl gradient of 0.15 - 3M.
  • the crude conjugate was applied to the column [S-Sepharose Fast Flow] on 0.15M NaCl and eluted with a 0.15 - 3M linear gradient of NaCl.
  • the conjugate peak eluted at approximately 1M NaCl,
  • the protein peak was pooled and dialyzed against 0.02M HEPES buffer, 0.15M sodium chloride buffer, pH 7.2.
  • the derivatized antibody was reduced with cysteine and purified by gel filtration using 25mM HEPES, 0.5M NaCl, pH 7.9, as running buffer. The fractions were pooled and the concentration of thiol groups determined using an Ellmanps test. A 2 molar excess of maleimido-b-Alanyl-NBC2- (S-acetamid ⁇ mcthyl-Cys) -COOH (5mg/ml) were added to the antibody-PDPH and the solution was left for 16h at room temperature. The concentration of NaCl was adjusted to 0.15M before the antibody*NBC2 conjugate was purified by cation exchange on ⁇ f sepharose using a NaU gradient of 0.15 - 3M. The protein peak was pooled and the pool sterile filtered and the conjugate stored at 4°C.
  • the peptide may then be u ated with 2-bromoaceric acid N-hydro.xysuccinimidyl ester water to give the N-(2-bromoacetyl)-peptide.
  • N-(2-bromoacetyl)-peptide followeded by reaction of a solution of the peptide in water with a large excess of hydrazine in the presence of EDC-I to yield the N- (2-bromoacetyl) -penta-glutary I -hydrazide derivative of the peptide. 5. Synthesis of a Clustered Monoclonal Antibody Ligand and its Conjugation to
  • the concentration of NaCl- was adjusted to 0.15M before the clustered antibody-NEC2 conjugate was purified by cation-exchange on SP Sephaiose using a NaCl gradient of 0115 - 3M.
  • the protein peak was pooled and the pool sterile filtered and the conjugate stored at 4°C.
  • MPIC 4(a-D-mannopyranosyloxy)phenylisothiocyanate
  • Mannosylated NBCl fractions were pooled and dialysed for 20h against 25mM HEPES, 0.15M NaCl, pH 7.5. It was estimated that 30% of the lysyl residues of NBCl were mannosylated using this method. 2. Preparation of Mannosylated Lvsine Dendrimer
  • the 2nd generation lysine dendrimer [Den2: (Lys)3-Gly-GI -Tyr-CysJ was synthesised using a Miliipore 9050 Plus Pepsynthesiser running in extended cycle mode (lh couplings) with TBTU / N-ethyldiisopropylamine activation and deprotection with 20? ⁇ Piperidine in dimethylformamide.
  • die peptide was synthesised sequentially using the following amino acid derivatives; Fmoc-0(tert-bu ⁇ yl>ty ⁇ osine; Fmoc-glycine, di-Fmoc-lysine and Fmoc-Ne-tert-butoxycarbonyl lysine. After the final coupling had finished the N-terminal Fmoc groups were removed using 20% piperidine in dimethylformamide.
  • the peptide was cleaved from the dried resin using reagent K (TFA, water, phenoLthioanisole.l,2-e ianeditbiol; 82.5:5:5:5:2.5) and purified by ion-exchange (SP-sepharose)and reverse phase hplc.
  • reagent K water, phenoLthioanisole.l,2-e ianeditbiol; 82.5:5:5:5:2.5
  • SP-sepharose reverse phase hplc.
  • the peptide solution was then added to 95mg 4-(a-D-maimopyranosyloxy)-phenylisothiocyanate dissolved in 1ml of the acetoni rile/borate solution.
  • the mannosylation was left to proceed at 37 5 C for I6h.
  • the thiol of the mannosylated peptide was reduced by adding 20mg solid dithiothreitol.
  • the peptide was then purified by high resolution gel filtration.
  • the mannosylated peptide was deterrain ⁇ d to possess, on average, between 3 and 4 mannose groups per peptide molecule.
  • a maleimide function was introduced into NBC12 by addition of 4.3mg (I2.2mmol) 4(-4-maleimidophenyl)butyric acid hydrazide.
  • O.IM sodium acetate pH 4.6, containing 20% acetonitrile.
  • After 15h incubation at room temperature the hydrazone was purified by gel filtration in 1% acetic acid, pH 4.7. The peptide solution was lyophilised.
  • Man4Den5-NBC12 is a mannosylated dendrimer derivative of NBC- 12, similar in structure of Man4De ⁇ 2-NBC121. In this case however the mannosyl side chains are introduced using solid phase peptide synthesis coupling methods with the tetra-o-acetyl-mannose-a-Fmoc- Serine derivative.
  • the peptide dendrimers were synthesised using a Miliipore 9050 Plus Pepsynthesizer as described above except that Fmoc-Ne-tert-butoxycarbonyl lysine 3 replaced by di-Fmoc-lysine.
  • the four F oc-protected amine groups were deprotected using 20% piperidine in dimethylformamide.
  • the free amines were then reacted with a ten-a-O-Ac-mann-a-Fmoc-serinc residue (in 16-fold molar excess of peptide) with TBTU/N-ethyldiisopropylamine as activating agent.
  • N-terminal Fmoc groups were removed using 2% D3U in dimethylformamide containing 2% piperidine.
  • glycopeptides were cleaved from the resin by adding an excess of 95% TFA for 2h at room temperature.
  • the glycopeptide was precipitated in 10 volumes of ether, the ether discarded after centrifugation and purified by hplc.
  • the peptides were treated withNaOMe/MeOH before a final purification by HPLC.
  • the glycopeptide, Man4Den5 was coupled to NBC 12 via a maleimide-hydrazide bifunctionai crosslinker, as described above.
  • the glycopeptide was synthesised with a C-terminal hydrazide group by using a hydroxymethylbenzoic acid (HMB A) resin linker and cleavage method, as described in "synthesis of Man4Den4".
  • HMB A hydroxymethylbenzoic acid
  • Such hydrazide derivatives can be used to couple the Man peptide directly to oxidised NBC 12, as described for the synthesis of Man4Den4-NBC12.
  • a 6 kilobase (kb) plasmid DNA (pRSVLuc) was biotinylated using photoactivatable biotin (Pierce and arriner) as per manufacturer's instructions. Estimated biotin level is one biotin every 100-400 bases.
  • a sensor chip with immobilized streptavidin was conditioned following manufacturer's instructions, i.e., three one-minute injections of 1M NaCl: 50mM NaOH followed by one Imin injection of 0.5% SDS.
  • Flow rate on the BIAcore sensing device was 20 ⁇ l per min, running buffer was phosphate buffered saline (PBS).
  • Flow path was flow cells 1 and 2. The flow path was switched to flow cell 2 only and the flow rate was reduced to lO ⁇ l per minute.
  • Biotinylated plasmid at a concentration of 200 ⁇ l ml in 0.5M NaCl was injected for 3 mins. This resulted in an increase of 614 RU after the injection.
  • Phosphate buffered saline was used as running buffer at a flow rate of 20 ⁇ l per minute.
  • the flow path was over flow cells 1 and 2.
  • Flow cell 1 was used as a reference cell. The experiments were carried out at a temperature of 25 ⁇ C.
  • Peptides were injected at various concentrations for three minutes. The injection was followed by a 5min dissociation phase before the injection port was washed. The sensor chip was regenerated by the injecxion of 2M NaCl for Imin. If necessary, this injection was repeated to bring the response back down to baseline before the next peptide sample was injected.
  • Example 7 Preparation of Complexes for In Vivo Delivery
  • Peptide or conjgate ⁇ g/ ⁇ g ⁇ lipidated peptide ⁇ g/ ⁇ g ( ⁇ g ⁇ g referring to ⁇ g peptide per ⁇ g DNA); DNA concentration; NaCl concentration.
  • Buffer is normally 25mM phosphate pH 7.4, but can be 25mM Hepes pH 7.4.
  • the amount of complex required for the experiment is calculated in terms of volume and total mass of DNA. The volumes of the different components required are calculated.
  • NBC peptide or conjugate at lOmg/ml or lmg/ml
  • Lipidated peptide at lOmg/ml or lmg/ml
  • Phosphate buffer at 0.5M
  • NaCl at 5M
  • DNA - concentration is variable, normally 1 - lOmg/ml.
  • the total volume is made up with water.
  • mice The ability to transfect tumor cells in vivo in mice was tested using plasmid nucleic acid carrying the nitroreductase reporter gene condensed on each of the following peptides: NBCl 5, NBC8, NBC9, NBC 11 , NBC7.
  • DBA2 - male mice aged 5 - 10 weeks were injected subcutaneousiy in each flank with a squamous epithelial syngenic tumour cell line KLN-205.
  • the cell fire was grown up in 175ml T-flasks in standard EMEM medium containing 10% foetal calf serum.
  • the cells reached a density of 4 x 10* they were suspended by a standard trypsinization technique, washed in PBS and diluted to a final concentration of 5 x 10 '9 cells 50 ⁇ l.
  • mice were anaesthetized before injection using the following preparation:
  • the anaesthetic used is Enflurane (Alynane; Anaquest, Ltd.. W ⁇ ndlesham. Surrey, UK); Ketamine (10% Ketaset. Willows Francis Veterinary, Cramley, West Wales, UK); Rompun (2%, Bayer UK, Ltd., Suffolk, UK).
  • Ketamine 10% Ketaset. Willows Francis Veterinary, Cramley, West Wales, UK
  • Rompun 2%, Bayer UK, Ltd., Suffolk, UK.
  • 10ml anaesthetic mixture 8.6ml PBS, 1 l Ketamine, 0.5ml Rompun.
  • Each animal was injected intraperitoneaUy with 200 ⁇ l animal wing I ml syringe wi h 25 gauge needle.
  • mice When anaesthetized the mice were injected with the cell suspension as follows: 2 c ⁇ r areas on both flanks of the animal were shaved and 50 ⁇ l of the cell suspension injected subcutaneously into each flank using a 1ml syringe with a 25 gauge needle. The mice were then kept until the tumours were palpable.
  • tumours appear palpable, growth was monitored on a daily basis and sized documented via their longest dimension. Once tumours reach desired size (5 - 8-mm is acceptable) then can be injected.
  • mice were first anaesthetized as follows:
  • the anaesthetic was Enflurane fAlynane; .Anaquest, Ltd., W ⁇ dlesiiam, Surrey UK); Ketamine (10% Ketaset, Willows Francis Veterinary. Cramley, West Wales, UK): Rompun (2%, Bayer UK. Ltd. , Suffolk, UK).
  • Ketamine 10% Ketaset, Willows Francis Veterinary. Cramley, West Wales, UK
  • Rompun 2%, Bayer UK. Ltd. , Suffolk, UK.
  • lO i anaesthetic mixture 8.5ml PBS; 1ml Ketamine; 0.5ml Rompun.
  • Each animal was injected intia-peritoneally with 200 ⁇ l/animal using lml syringe with 25 gauge needle.
  • the tumour is manipulated between the fingers lifting it slightly away from the -tn ⁇ mafs body and at the same time appiy minimum pressure to the tumour.
  • the formulated delivery vehicle a plasmid containing the E. coli nitroreductase gene condensed on a. peptide at a ratio of O.i ⁇ g peptide/ ⁇ g DNA
  • Daily monitoring is continued until the experiment is terminated,
  • Sections of frozen tumour tissue are prepared using standard methods and a Cryostat and air dried. Sections are fixed with freshly prepared 4% PF A PBS for 30mins at room temperature and then washed twice with 0.01% Tween-20/PBS 5mins each wash. Sections are then immersed in 025% hydrogen peroxide PBS, at room temperature (20C) for 30mins, and then washed three times in 0.01% Tween 20/PBS far 5mins each wash. Sections are then permeabilised by treatment with ice cold 0.1% TRITON X-100 for 5mins, followed by a further two washes in 0.01% Tween-20/PBS 5mins each wash.
  • the sections are then blocked with 5% NGS/PBS 60mins at room temperature in a wetbox. After decanting the blocking solution, 0.5ml diluted antiserum/slide (1 :400 in 2.5% NGS) is added and the sections incubated for 60mins at room temperature. Slides are then washed three times in 0.01% Tween-20/PBS Smirts each wash. Secondary antibody (0,5ml) from ABC Elite Vector stain kit is added as follows and incubated 30mins at room temperature in a wetbox.
  • the antibody solution was as follows: PBS (lOmls), NGS (0.15ml), antibody (0.05ml).
  • the staining solution was prepared 30mins prior to use. and included: 5mls PBS. 2 drops solution A, 2 drops solution B.
  • Sections are washed three times in 0.01% Tween-20 PBS 5mins each.wash followed by addition of 0.5ml Vector stain per slide and incubated 30mins room temperature in a wetbox. The slides are then washed three times in PBS, 5mins each wash.
  • AEC substrate from Vector AEC Kit. Vector Labs
  • AEC substrate from Vector AEC Kit. Vector Labs
  • transfection efficiency was determined by transfecting tumor cells ex vivo, injecting the transfected cells IP into the animal, and determining transfection efficiency, a ⁇ follows.
  • mice were DBA2 - Male, age 5 - 1-0 weeks.
  • Culture of the adherent cell line KLN-205 (ATCC) were grown in 10% FCS in EMEM - 1% NEAA (Life Technologies, Gaithersburg, Maryland) and subcultured using a standard trypsin- EDTA treatment prior to transfer, The approximate number of cells from T175 is 4 x 10 4 . Two subculture steps were performed.
  • mice were washed three times in PBS. The final concentration of cells was 1 x lOVlOO ⁇ l (animal), i.e. 5 x 10 s /50 ⁇ l - 50 ⁇ l injected-'flank. Mice were anaesthetized with Enflurane (Alynane; Anaquest, Ltd., Windlesbam, Surrey, UK); Ketamine (10% Ketasst, Willows Francis Veterinary, Cramley. West Wales, UK); and Rompun (2%, Bayer UK, Ltd., Suffolk, UK). For each 1 ml anaesthetic mixture: 8.5ml was physiological saline; lml Ketamine; and 0.5ml Rompun.
  • Animals were anaesthetized by injectiing 200 ⁇ l anirnal interperitoneally using lml syringe with 25G needle. Animals were fully anaesthetized in approximately 5mins.
  • a standard ELISA was performed using Amersham antisera. That is, the antigen solution was prepared in c ⁇ anng buffer, l ne amount should be ⁇ etermine ⁇ by cnequer-boar ⁇ , nowever in the range of 50ng/100 ⁇ l coating buffer is a typical amount to use.
  • wells are filled with lO ⁇ l of antigen/coating buffer. The plate is sealed with parafilm to avoid evaporation. The plate is incubated overnight at 4 ⁇ C or 37 °C 2h. A wash is then performed with PBS Tween. lOO ⁇ l of primary antibody is applied to a first column of wells, and the remainder of wells are double diluted across the plate, leave the last column as negative control i.e. PBS/Tween. The plate is then incubated.
  • Fig. 6 shows the frequency of transfection of tumor tissue cells obtained with complexes of NBCl 5 and NBC9 were used.
  • the highest number of transfected tumor cells was obtained using peptide- nucleic acid complexes containing the NBC15 peptide (i.e., most efficient transfection) and the fewest number of transfected tumor cells was obtained using complexes containing a poly-lysine peptide (i.e., least efficient transfection).
  • Figure 6(a) shows the transfection pattern obtained when tumours are injected with plasmid pTX 118 formulated with 0.5 ⁇ g NBC 1 peptide/ ⁇ g DNA.
  • the tumour is a murine syngenic squa ous carcinoma produced by subcutaneous injection of KLN205 cells.
  • the tumours were approximately 4mm in diameter they were injected with 20 ⁇ l of plasmid DNA formulated with the NBC 15 peptide.
  • the tumours were excised and sectioned by standard bistological techniques and immunostained for expression of the transgene (E. coti nitroreductase).
  • the figure shows large numbers of cells in the tumour expressing the transgene (stained red).
  • Figure 6(b) shows a similar experiment. In this case the tumours were mjected with the same plasmid formulated in 0.5 ⁇ g NBC9 peptide/ ⁇ g DNA. Although transfection is evident the level of transfection is noticeably lov/er.
  • the relative ability of the peptides in this study to transfect cells can be summarized in a ranking based on the numbers of transfected tumor cells obtained as an indication of transfection efficiency, i.e., from most efficient transfection to the least efficient tiansfection: NBCl 5 > NBC8, NBC9, NBCl 1 > NBC7 >» poly-lysine.
  • pRSVLuc DNA at a concentration of 1 OO ⁇ g/ml was condensed using peptide at a ratio of 2 ⁇ g/ ⁇ g plasmid DNA as described herein with the following deviation: prior to addition to the cells (Jurkat cell line) the complexes were diluted to 20 ⁇ g/mllwith 10% PEG 10 000; 37raM sodium chloride; 25mM sodium phosphate, pH 7.4. The cells were assayed for luciferase after 24h.
  • the relative transfection efficiency of individual NBC peptides and a polylysine peptide is shown in Fig. 7.
  • Fig. 7 of describes transfection of a cell line with a luciferase plasmid condensed with different cationic peptides and shows a rank in order of relative transfection efficiency of: (most efficient) NBCl 1 * NBC 13 > NBC8 » NBC 15 >» polylysine (least efficient).

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Abstract

L'invention concerne des méthodes de criblage de peptides permettant, dans un système de fourniture de gènes, une transfection optimale de cellules fondée sur des paramètres cinétiques de l'interaction bimoléculaire peptide-acide nucléique.
EP97951337A 1996-12-23 1997-12-23 Optimisation de fourniture de genes et de systemes de fourniture de genes Withdrawn EP0946878A1 (fr)

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GBGB9626992.3A GB9626992D0 (en) 1996-12-27 1996-12-27 Optimization of gene delivery and gene delivery systems
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PCT/GB1997/003523 WO1998028626A1 (fr) 1996-12-23 1997-12-23 Optimisation de fourniture de genes et de systemes de fourniture de genes

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