Safety of the use of gold nanoparticles conjugated with proinsulin peptide and administered by hollow microneedles as an immunotherapy in type 1 diabetes.

Antigen-specific immunotherapy is an immunomodulatory strategy for autoimmune diseases, such as type 1 diabetes, in which patients are treated with autoantigens to promote immune tolerance, stop autoimmune ß-cell destruction and prevent permanent dependence on exogenous insulin. In this study, human proinsulin peptide C19-A3 (known for its positive safety profile) was conjugated to ultrasmall gold nanoparticles (GNPs), an attractive drug delivery platform due to the potential anti-inflammatory properties of gold. We hypothesised that microneedle intradermal delivery of C19-A3 GNP may improve peptide pharmacokinetics and induce tolerogenic immunomodulation and proceeded to evaluate its safety and feasibility in a first-in-human trial. Allowing for the limitation of the small number of participants, intradermal administration of C19-A3 GNP appears safe and well tolerated in participants with type 1 diabetes. The associated prolonged skin retention of C19-A3 GNP after intradermal administration offers a number of possibilities to enhance its tolerogenic potential, which should be explored in future studies.

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.

Topical propranolol for infantile haemangiomas: a systematic review.

Infantile haemangiomas are the most common tumour of infancy. Whilst the majority are left untreated to involute spontaneously, residual skin changes commonly occur, particularly in superficial haemangiomas. The current first-line treatment for problematic lesions is oral propranolol; however due to the risk of systemic adverse effects, the use of off-label topical preparations has recently been investigated. Our systematic review was conducted in accordance with PRISMA guidelines. Four databases were searched to identify original articles evaluating the use of topical propranolol as the primary therapy for infantile haemangiomas. Twelve articles with a total of 597 patients and 632 haemangiomas were included. Three topical propranolol preparations were used, creams, ointments and gels and were all prepared by local pharmaceutical laboratories. The concentration of propranolol ranged from 0.5% to 5%. Treatment duration ranged from two weeks to 16.5 months. Overall, 90% of lesions improved following the initiation of topical propranolol. A good or excellent response, defined as a reduction in the size of at least 50%, was seen in 59% of lesions. Earlier initiation of treatment (less than 3 months of age) was associated with improved outcomes. No systemic adverse effects were reported. Minor local reactions were seen in 1.3% of patients. Topical propranolol is safer than oral propranolol, though may be less effective. Topical propranolol may be more suitable for patients with small, superficial haemangiomas at risk of cosmetic sequelae, where the cosmetic or symptomatic impact does not warrant oral propranolol treatment.

Minimally invasive and targeted therapeutic cell delivery to the skin using microneedle devices.

BACKGROUND: Translation of cell therapies to the clinic is accompanied by numerous challenges, including controlled and targeted delivery of the cells to their site of action, without compromising cell viability and functionality. OBJECTIVES: To explore the use of hollow microneedle devices (to date only used for the delivery of drugs and vaccines into the skin and for the extraction of biological fluids) to deliver cells into skin in a minimally invasive, user-friendly and targeted fashion. METHODS: Melanocyte, keratinocyte and mixed epidermal cell suspensions were passed through various types of microneedles and subsequently delivered into the skin. RESULTS: Cell viability and functionality are maintained after injection through hollow microneedles with a bore size ≥ 75 µm. Healthy cells are delivered into the skin at clinically relevant depths. CONCLUSIONS: Hollow microneedles provide an innovative and minimally invasive method for delivering functional cells into the skin. Microneedle cell delivery represents a potential new treatment option for cell therapy approaches including skin repigmentation, wound repair, scar and burn remodelling, immune therapies and cancer vaccines.

Hydrodynamic gene delivery in human skin using a hollow microneedle device.

Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVß and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50µL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVß reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery.

Pocketed microneedles for rapid delivery of a liquid-state botulinum toxin A formulation into human skin.

Botulinum toxin A (BT) is used therapeutically for the treatment of primary focal hyperhidrosis, a chronic debilitating condition characterised by over-activity of the eccrine sweat glands. Systemic toxicity concerns require BT to be administered by local injection, which in the case of hyperhidrosis means multiple painful intradermal injections by a skilled clinician at 6-monthly intervals. This study investigates the potential of a liquid-loaded pocketed microneedle device to deliver botulinum toxin A into the human dermis with the aim of reducing patient pain, improving therapeutic targeting and simplifying the administration procedure. Initially, ß-galactosidase was employed as a detectable model for BT to (i) visualise liquid loading of the microneedles, (ii) determine residence time of a liquid formulation on the device and (iii) quantify loaded doses. An array of five stainless steel pocketed microneedles was shown to possess sufficient capacity to deliver therapeutic doses of the potent BT protein. Microneedle-mediated intradermal delivery of ß-galactosidase and formaldehyde-inactivated botulinum toxoid revealed effective deposition and subsequent diffusion within the dermis. This study is the first to characterise pocketed microneedle delivery of a liquid formulation into human skin and illustrates the potential of such systems for the cutaneous administration of potent proteins such as BT. A clinically appropriate microneedle delivery system for BT could have a significant impact in both the medical and cosmetic industries.

Quantifying the mechanical properties of human skin to optimise future microneedle device design.

Microneedle devices are a promising minimally invasive means of delivering drugs/vaccines across or into the skin. However, there is currently a diversity of microneedle designs and application methods that have, primarily, been intuitively developed by the research community. To enable the rational design of optimised microneedle devices, a greater understanding of human skin biomechanics under small deformations is required. This study aims to develop a representative stratified model of human skin, informed by in vivo data. A multilayer finite element model incorporating the epidermis, dermis and hypodermis was established. This was correlated with a series of in-vivo indentation measurements, and the Ogden material coefficients were optimised using a material parameter extraction algorithm. The finite element simulation was subsequently used to model microneedle application to human skin before penetration and was validated by comparing these predictions with the in-vivo measurements. Our model has provided an excellent tool to predict micron-scale human skin deformation in vivo and is currently being used to inform optimised microneedle designs.

Low temperature fabrication of biodegradable sugar glass microneedles for transdermal drug delivery applications.

Transdermal drug delivery is limited by the barrier properties of the outer skin layer. Microneedles (MNs) effectively circumvent the skin barrier to offer this route as a potential alternative to oral and parenteral delivery of therapeutics. Biodegradable microneedles offer particular advantages however processing commonly requires elevated temperatures that may adversely affect heat-labile molecules and macromolecules. In this study, solid amorphous sugar glasses containing low residual quantities of water were created by dehydration of trehalose and sucrose sugar combination solutions. Biodegradable sugar glass MNs were fabricated following optimisation of a simple and novel low temperature vacuum deposition micromoulding methodology. These had absolute morphological fidelity to silicon master structures and demonstrated sufficient structural rigidity to efficiently penetrate excised human breast skin. Sugar glass MNs incorporating a marker compound dissolved rapidly and completely in situ releasing dye into deeper skin layers. The biological activity of a model macromolecule was partially retained over extended storage following incorporation into sugar glass. This is the first demonstration that MNs created from amorphous sugar glasses can be used for incorporating and delivering molecules, and potentially biologically active macromolecules, via the transdermal route.

Clinical administration of microneedles: skin puncture, pain and sensation.

Injections using hypodermic needles cause pain, discomfort, localised trauma and apprehension. Additionally, careful use and disposal of needles is required to avoid transmission of blood-borne pathogens. As an alternative, microneedles can facilitate drug delivery without significantly impacting on pain receptors or blood vessels that reside beneath the skin outer layers. In this study we aim to determine the pain and sensory response to the application of wet-etch silicon microneedles, when used in such a way as to reliably penetrate skin, and provide a preliminary indication of how skin responds to microneedle injury with time. Twelve subjects received single-blinded insertions of a 25-G hypodermic needle and two microneedle arrays (36 needles of 180 and 280 mum height). The optimal method for microneedle application was determined in a pilot study. Pain intensity was scored using a visual analogue scale (VAS) and sensory perception determined using an adapted McGill Pain Questionnaire Short Form. Skin penetration was determined by external staining and measurement of trans-epidermal water loss (TEWL). Mean VAS scores, verbal descriptions and questionnaire responses showed that the 180 and 280 mum microneedles caused significantly less pain and discomforting sensation in participants than the hypodermic needle. Methylene blue staining and TEWL analysis confirmed that microchannels were formed in the skin following microneedle application. Evidence of microchannel repair and resealing was apparent at 8-24 h post-application. In summary, this study shows that pyramidal wet-etch microneedles can penetrate human skin with minimal pain and sensory discomfort, creating transient pathways for potential drug, vaccine and DNA delivery.

A comparison of the puncturing properties of gelatin and hypromellose capsules for use in dry powder inhalers.

This study investigates capsule puncture in dry powder inhalers. Gelatin and hydroxypropylmethyl cellulose (HPMC) capsules (stored at 11 and 33% relative humidities) were punctured using a pin from a Foradil inhaler, with insertion force measurement via an Instron tester. In HPMC capsules, the force after capsule puncture reduced by half and then increased to a second maximum as the pin shaft entered the hole. In gelatin capsules, the postpuncture force reduced to zero, indicating shell flaps losing contact with the pin. At lower moisture contents, both capsules were less flexible. This provides a tool to measure the shell properties of inhalation capsules.

The use of absorption enhancers to enhance the dispersibility of spray-dried powders for pulmonary gene therapy.

BACKGROUND: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. METHODS: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. RESULTS: Spray-dried powder containing dimethyl-beta-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. CONCLUSIONS: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model.

The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy.

BACKGROUND: Pulmonary delivery of gene therapy offers the potential for the treatment of a range of lung conditions, including cystic fibrosis, asthma and lung cancer. Spray-drying may be used to prepare dry powders for inhalation; however, aerosolisation of such powders is limited, resulting in poor lung deposition and biological functionality. In this study, we examine the use of amino acids (arginine, aspartic acid, threonine, phenylalanine) to enhance the aerosolisation of spray-dried powders containing model non-viral gene vectors. METHODS: Lipid/polycation/pDNA (LPD) vectors, in the presence or absence of amino acids, were dispersed in lactose solutions, and spray-dried to produce appropriately sized dry powders. Scanning electron microscopy and laser diffraction were used to determine particle morphology and diameter, respectively. Gel electrophoresis was used to examine the influence of amino acids on the structural integrity of the LPD complex. In vitro cell (A549) transfection was used to determine the biological functionality of the dry powders, and the in vitro aerosolisation performance was assessed using a multistage liquid impinger (MSLI). RESULTS: Both gel electrophoresis and in vitro cell transfection indicated that certain amino acids (aspartic acid, threonine) can adversely affect the integrity and biological functionality of the LPD complex. All amino acids significantly increased the aerosolisation of the powder, with the arginine and phenylalanine powders showing optimal deposition in the lower stages of the MSLI. CONCLUSIONS: Amino acids can be used to enhance the aerosolisation of spray-dried powders for respiratory gene delivery, allowing the development of stable and viable formulations for pulmonary gene therapy.

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying.

BACKGROUND: Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze-drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non-viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze-drying and spray-drying, a one-step process for producing dry and respirable powders, as methods for preparing non-viral respiratory gene delivery systems. METHODS: Lipid:polycation:pDNA (LPD) vectors comprising 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP-N1 in 3% lactose solution were either snap-frozen and lyophilised or spray-dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence. RESULTS: Lyophilisation produced large, irregularly shaped particles prior to (mean diameter approximately 21 microm) and following (mean diameter approximately 18 microm) coarse grinding. Spray-drying produced uniformly shaped spherical particles (mean diameter approximately 4 microm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray-dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage. CONCLUSIONS: Spray-drying has utility for producing stable, efficient and potentially respirable non-viral dry powder systems for respiratory gene delivery.

Statistical modelling of the formulation variables in non-viral gene delivery systems.

Traditionally, optimisation of a gene delivery formulation utilises a study design that involves altering only one formulation variable at any one time whilst keeping the other variables constant. As gene delivery formulations become more complex, e.g. to include multiple cellular and sub-cellular targeting elements, there will be an increasing requirement to generate and analyse data more efficiently and allow examination of the interaction between variables. This study aims to demonstrate the utility of multifactorial design, specifically a Central Composite Design, in modelling the responses size, zeta potential and in vitro transfection efficiency of some prototypic non-viral gene delivery vectors. i.e. cationic liposome-pDNA complexes, and extending the application of the design strategy to more complex vectors, i.e. tri-component lipid:polycation:DNA (LPD). The modelled predictions of how the above responses change as a function of formulation show consistency with an extensive literature base of data obtained using more traditional approaches, and highlight the robustness and utility of the Central Composite Design in examining key formulation variables in non-viral gene delivery systems. The approach should be further developed to maximise the predictive impact of data across the full range of pharmaceutical sciences.

Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes.

The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid-DNA complexes. Complexes prepared from the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass (w/w) ratios of 12:1, 6:1 and 3:1, and complexes prepared from DOTAP, the polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145+/-3 nm pre-nebulisation vs. 142+/-2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102+/-1 nm pre-nebulisation vs. 113+/-2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.

Gene expression in an intact ex-vivo skin tissue model following percutaneous delivery of cationic liposome-plasmid DNA complexes.

The skin represents an attractive site for the localised gene therapy of dermatological pathologies and as a potential antigen bioreactor following transdermal delivery. Potential also exists for the gene therapy of skin as a cosmetic intervention. The most exploited non-viral gene delivery system involves the complexation of cationic liposomes with plasmid DNA (pDNA) to form lipid:pDNA vectors that protect the DNA from nuclease-mediated degradation and improve transgene-cell interactions. Despite numerous studies examining the potential for these vectors in delivering genes to a variety of keratinocyte models, investigations into the topical application of such complexes to intact skin tissue is limited. This ex-vivo study, conducted with intact skin tissue derived from hairless mice, provides quantitative confirmation that topical administration of cationic lipid:pDNA complexes can mediate uptake and expression of reporter pDNA (33-fold higher compared with control) in viable epidermal tissue. The ex-vivo study design provides for intact skin tissue that has not been subjected to depilatory procedures of potential detriment to stratum corneum barrier function, and can be utilised for the quantitative and efficient examination of a potentially wide range of non-viral gene vectors designed for epidermal expression.

Physico-chemical characterisation and transfection efficiency of lipid-based gene delivery complexes.

Cationic liposomes spontaneously interact with negatively charged plasmid DNA to form a transfection competent complex capable of promoting the expression of a therapeutic gene. This work aims to improve the understanding of the poorly defined mechanisms and structural rearrangements associated with the lipid-DNA interaction. Specifically, dimethyl dioctadecylammonium bromide (DDAB):dioleoyl phosphatidylethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) liposomes were mixed with a reporter plasmid (pADbeta or pCMVbeta) to form lipid-DNA complexes. The size and charge characteristics of the complexes as determined by photon correlation spectroscopy and microelectrophoresis were found to be dependent on the lipid:DNA ratio, with both DDAB:DOPE-DNA and DOTAP-DNA complexes aggregating at around neutral zeta potential. Negative stain transmission electron microscopy demonstrated at least three distinct complex structures being formed at the same DOTAP:DNA ratio. We postulate that two of these aggregates are structural moieties involved in the formation of the efficient transfection particle. Gel electrophoresis was used to determine the efficiency and extent of lipid-DNA complex formation. Results showed that only DOTAP liposomes were capable of preventing ethidium bromide intercalation with DNA and protecting the enclosed plasmid from nuclease digestion. When a range of lipid-DNA complexes were transfected into in vitro cell lines, the efficiency of reporter gene (beta-galactosidase) expression was found to depend on the type of liposome used in the complex, the ratio of lipid:DNA and the transfected cell line. Our results challenge the requirement for DOPE to be included in the formulation of cationic lipid vectors, especially in the case of DOTAP containing liposomes.

Production of null mutants in the major intestinal esterase gene (ges-1) of the nematode Caenorhabditis elegans.

The ges-1 gene of the nematode Caenorhabditis elegans codes for a nonspecific carboxylesterase that is expressed only in the intestinal lineage. This esterase has turned out to be a convenient biochemical marker for lineage-specific differentiation. In the present paper, we describe the production of several C. elegans strains that lack detectable activity of the ges-1 esterase. To isolate these ges-1 null strains, we first produced a strain of hermaphrodites in which the wild-type copy of the ges-1 gene was stably balanced over a previously isolated isoelectric focusing allele, ges-1(ca6); this parental strain was then mutagenized with EMS and isoelectric focusing gels were used to identify progeny populations that lacked either ges-1(+) or ges-1(ca6) esterase activity. This method is a straightforward and general approach to obtaining null mutations in any gene that has a biochemical or immunological assay. The ges-1 gene is not essential to worm survival, development or reproduction. Furthermore, lack of the ges-1 product has no obvious effect on the ability of worms (containing either normal or greatly reduced levels of acetylcholinesterases) to survive exposure to esterase inhibitors. The ges-1 gene product provides roughly half of the total esterase activity measured in crude extracts of L1 larvae or mixed worm populations. However, histochemical staining of individual ges-1(0) embryos shows that the ges-1 esterase is the first and essentially the only esterase to be produced during embryonic development, from the midproliferation phase up to at least the twofold stage of morphogenesis. These ges-1(0) strains now allow us to investigate the developmental control of the ges-1 gene by DNA-mediated transformation, in which the ges-1 gene acts as its own reporter.