WO2004069993A2

WO2004069993A2 - Non-touch zona pellucida microdrilling of a single - cell embryo with a 1.48 um laser beam for introduction of retrovirus

Info

Publication number: WO2004069993A2
Application number: PCT/EP2004/001101
Authority: WO
Inventors: Alfred Senn; Thierry Pedrazzini
Original assignee: Octax Microsciences Gmbh; Mtg Medical Technology Vertriebs Gmbh
Priority date: 2003-02-06
Filing date: 2004-02-06
Publication date: 2004-08-19
Also published as: EP1589811A2; WO2004069993A3

Abstract

The present invention concerns the use of a 1.48 µm laser to microdrill a hole in the zone pellucida of a single-cell embryo. Thereby, a retrovirus, especially a Lentivirus carrying a particular transgene can be introduced into said embryo.

Description

NON-TOUCH ZONA PE UCIDA MICRODRILLING OF OOCYTES WITH A 1.48 μm DIODE LASER BEAM FOR INTRODUCTION OF RETROVIRUS

The advent of transgenic technology some twenty years ago represented a major breakthrough in biomedical research, and has become an indispensable tool to biologists. Classical transgenesis in the purpose of generating genetically modified animals carrying copies of a given exogenous gene is currently performed by pronuclear injection. However, this technique remains relatively inefficient and impractical in most species. Recently, germline transmission of transgenes was obtained after delivery by retroviral vectors; lentiviral vectors were specifically used.

In Lois et al . , Science, Vol. 295., pp. 868-872, Feb.2002, the following is disclosed: Single-cell mouse embryos were infected in vi tro with recombinant lentiviral vectors to generate transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter. Eighty percent of founder mice carried at least one copy of the transgene, and 90% of these expressed GFP at high levels. Progeny inherited the transgene (s) and displayed green fluorescence. Mice generated using lentiviral vectors with muscle-specific and T lymphocyte- specific promoters expressed high levels of GFP only in the appropriate cell types.

Among other retroviruses which are useful as the delivery tools, lentiviruses are a class of retroviruses that cause chronic illnesses in the host organisms they infect . Among retroviruses, lentiviruses have the distinguishing property of being able to infect both dividing and nondividing cells, and this ability has led to their development as gene delivery vehicles. Because of their property to infect both dividing and non-dividing cells, they have been used widely as gene delivery tools. In one procedure, lentiviruses engineered to carry a transgene were injected into the perivitelline space of single cell mouse embryos. Embryos were implanted into pseudo-pregnant mothers, and carried to term. A large percentage of the resulting progeny was shown to have integrated the transgene. In an alternative procedure, the zona pellucida was removed and the denuded embryos were incubated with the lentiviral suspension for three days. Although both procedures were able to produce transgenic mice at a high rate, they remain technically quite demanding. Indeed, in the first alternative, injection is still required whereas long term culture of embryos is necessary in the second case.

Chemical (e.g. Gordon and Talansky, J. Exp. Zool . , 239, 347- 354, 1986; Garrisi et^'al., Fertil . Steril, 54, 671-677, 1990), mechanical (e.g. Depypere et al . , Hum.Reprod. Fertil, 84, 205- 211, 1988); or laser methods (e.g. Feichtinger et al . , Lancet, 339, 811, 1992) have been used to produce holes in the zona pellucida of mammalian oocytes in order to improve fertilization (Gordon, Ann.N.Y.Acad. Sci . , 541, 601-613, 1988; el Danasouri et al . , Hum.Reprod., 8, 464-466, 1993; Ng et al . , J. ssist .Reprod. Genet . , 10 (Suppl.), 204, 1993; Enginsu et al . , Hum. Reprod., 10, 1761-1764, 1995), facilitate blastocyst hatching (e.g. Cohen et al . , Hum. Reprod., 5, 7- 13, 1990;) or perform blastomere biopsy (Tarin and Handyside, Fertil. Steril. , 59, 943-952, 1993). Recently, an infrared 1.48 μm diode laser light, focused through a microscope objective, was shown to allow rapid, easy and non-touch microdrilling of mouse and human zona pellucida while maintaining a high degree of accuracy under conventional culture conditions (Rink et al . , Proceedings SPIE, 2134A, 412-422, 1994) . The drilling effect was shown to be due to a highly localized heat-dependent disruption of the zona pellucida glycoprotein matrix (Rink et al . , Lasers Surg. Med., 18, 52-62, 1996), a phenomenon markedly different from the photo-ablation induced by wavelengths close to the UV region (Neev et al . , J. Assist. Reprod. Genet., 9, 513-523, 1992, Hum. Reprod., 8, 939-944, 1993). Embryos could be maintained in their usual culture dish and medium during the drilling process without requiring special quartz optical equipment as for UV lasers (Schύtze et al . , Fertil . Steril . , 61, 783-786, 1994), a change of medium (Blanchet et al . , Fertil. Steril., 57, 1337-1341, 1992) or micromanipulators as for the 2.9 μm Erbium:YAG laser (Obruca et al . , Hum. Reprod.

9, 1723-1726, 1994) . Contrary to the detrimental effect on pre-compacted mouse embryos (Schiewe et al . , Hum. Reprod.,

10, 1821-1824, 1995) induced by the 308 nm xenon-chlorine excimer laser (Neev et al . , Hum. Reprod., 8, 939-944, 1993), the drilling process in the infrared region did not affect embryo survival in mice (Germond et al . , Fertil. Steril., 64, 604-611, 1995; Rink et al . , Lasers Surg.Med. , 18, 52-62, 1996) or in humans (Germond M. et al . , (1998) : Diode laser for assisted hatching. Photomedicine in Gynecology and Reproduction, Karger, Basel, 352-365, 2000) .

Some years ago, a method was published using the micro- drilling of the zona pellucida by way of a 1.48 μm diode laser to improve fertilization in case of zona hardening (M. Germond et al, "Improved fertilization and implantation rates after non-touch zona pellucida microdrilling of mouse oocytes with a 1.48 μm diode laser beam", in "Human Reproduction" vol.11, no.5, pp.1043-1048, 1996.) This method is safe, and does not produce any deleterious effects during development or in the adults. Microdrilling results in a partial denudation of the single-cell embryos. Depending on the irradiation time, the diameter of the drilled hole varied between 5-10 μm.

As shown above, a need remains to provide a procedure to produce transgenic animals at a high rate. Specifically, a need remains to provide a method without an injection and without the necessity for long-term culture of embryos.

Therefore, it is an object of the present invention to overcome the above-mentioned needs.

Summary of the Invention

The above object is overcome by the present invention by the use of a 1.48 μm laser to microdrill a hole in the zona pellucida of a single-cell embryo to introduce a retrovirus, preferably a lentivirus" carrying a particular transgene into said embryo.

Preferred embodiments of the invention are indicated in the dependent claims 2 to 5.

The present inventors used the above method to allow the retroviral vectors to be in contact with the plasma membrane of the embryos. Drilled fertilized oocytes were incubated for 2-24 hours preferably, for 3 to 5 , more preferred 4 hours with lentiviruses carrying a particular transgene. Because of the partial denudation, this makes the physical injection of the viral particles under the zona pellucida unnecessary. In addition, incubation time can be decreased to a few hours. Furthermore, the presence of the zona pellucida around the embryos make them stronger and more resistant to the whole procedure. Finally, the embryo can be transferred back into foster mothers the same day, and therefore the technique is less time consuming.

In a preferred embodiment the hole as microdrilled in the zona pellucida of a single-cell embryo is between 2 to 60 μm, preferably between 2 to 20 μm in diameter, more preferably 5 to 15 μm, even more preferred 5 - lOμm. Preferably, the zona pellucida of the single-cell embryo is exposed to one or up to 10 shots, preferably 1 to 5 shots, more preferred once or twice to 0.1 μs to 100 ms, preferably 0.1 to 15 ms of laser light, preferably 0.5 to 10 ms of laser light, more preferably 1 to 5 ms of laser light, depending on oocyte species and for laser power in focus of 20 - 500 mW, preferably 20 - 250 mW, more preferred 100 - 160 mW.

Increasing laser power leads to a decrease in the necessary irradiation time for a certain opening size. Using constant laser power the opening size increases with longer irradiation times, see e.g. Rink et al . 1996, supra.

The laser can be provided at any point of an optical system. Said optical system can in an exemplary embodiment be a microscope. In an exemplary embodiment, the laser is mounted directly in front of the objective (as seen from viewer to object) ; in another embodiment the laser is provided in front of or after the eye-piece or can replace e.g. the eye-piece or the CCD camera. The laser could in a further embodiment also be provided within the objective, e.g. in front of the last one to six lenses, preferably two to four lenses, of the objective. However, the actual arrangement of the laser is not restricted to the above embodiments. The laser can be provided together with any optical system, e.g. a microscope, in a preferred embodiment an inverse microscope.

The invention is further described with reference to the Figures which depict the following:

Fig. 1: The 1.48 μm diode laser assisted hatching unit.

Fig. 1A: The FERTILASE® system, with its control unit on the right, is attached to the fluorescence port at the back of the inverted microscope.

Fig. IB: The compact OCTAX™ system, with its octagonal laser in the back and the miniaturised video camera on the left. Fig. 2: Schematic of the 1,48 μm diode zona pellucida drilling arrangement. The fluorescence port of the inverted microscope is used to couple the surgical laser. The microscope objective is used to precisely focus the laser radiation onto the egg ZP.

Fig. 3 Zona pellucida drilling strategy. The focused laser beam is directed tangentially to the ZP to produce a trench.

Fig. 4 Human zygote drilled at the 2-pn stage on day 1. The laser drilled trench opens completely the ZP; it has been obtained by two consecutive 9 ms irradiation with the OCTAX laser system.

Fig. 5 Electron micrograph showing a trench drilled with the 1.48 μm diode laser system in a mouse zygote. Note the sharpness of the walls of the opening.

In the following the zona pellucida drilling is described in more detail :

Use of laser for zona pellucida drilling

In the present invention a 1.48 μm diode laser is used, preferably one of the type that was developed by the Institut d'Optique Appliquee (K. Rink and G. Delacretaz; at the Ecole Polytechnique Federale de Lausanne, Switzerland) in association with the Reproductive Medicine Unit (DGO; CHUV) (Rink et al . 1994 Supra; Rink et al . 1996 Supra) and commercialized as a functional unit (Fertilase^Θ; formerly Medical Technologies Montreux S.A., Clarens, Switzerland, now OCTAX) . Based on the experience with the Fertilase^®, the laser set-up has been optimized and commercialized a second time (OCTAX Laser Shot (TM) , OCTAX Microscience GmbH, Germany; http://www.octax.de) and has been supplemented with a miniature digital video camera and a computer software allowing laser control, image storage, database and analysis (Figure 1) Method for zona pellucida drilling

Zona pellucida (ZP) opening is performed according to the following procedure. The culture dish is placed on the displacement stage of the microscope (Figure 2) . As the laser wavelength is located in the infrared, drilling of the ZP can be performed directly in the culture dish while keeping the eggs in their original culture medium. With the help of the displacement stage, a region of the ZP, where the perivitelline space (PVS) is widest, is positioned at the location of the aiming spot . Opening is performed by exposing the ZP to the laser beam during 0.1 - 15 ms, preferably during 0.5-10 ms, more preferred 1-5 ms .

Several drilling strategies can be considered. If a single irradiation procedure is preferred, the hole size can be chosen by varying the^' irradiation time, typically a hole having a diameter of 20 μm is produced with a 12-30 ms irradiation time. Larger hole diameters are obtained by increasing the irradiation time. If the egg is placed tangentially to the diode laser beam a trench is induced in the ZP. By precisely positioning the laser focalization point with respect to the ZP width a complete opening or only a local thinning of the ZP can be generated at will (Figure 3) . If the egg is placed in such a way that the laser beam intersects the ZP in an area closer to the polar axis, while ensuring that the laser beam trajectory still remains in PVS, a cylindrical hole is formed. Direct interaction of the laser beam with the cytoplasm should be avoided. In Figure 4, a single irradiation has been applied onto the ZP of a two blastomere embryo, and the trench created has a diameter superior to the ZP thickness. However, a multiple irradiation procedure might be preferred by some operators in order to generate an opening with a specific shape. Because of the short irradiation time needed, no micromanipulators are necessary to stabilize the position of the eggs during the drilling procedure in the standard conditions. For the operator, ZP drilling is virtually instantaneous. Even if a multiple irradiation procedure has been chosen, no micromanipulators are needed, especially if the embryo has previously been placed in a culture medium microdrop under mineral oil .

An example of the striking quality and precision achieved by the 1.48 μm laser irradiation can be seen on an electron micrograph of a drilled mouse zygote in Figure 5.

The above technique can be used for all single-cell embryos comprising a zona pellucida. Particularly, it can be used for all mammals, including human and non-human embryos. In a more preferred embodiment, the embryo is a mouse or rat embryo .

In the following, the invention is described in more detail by way of an example which shall, however, not be construed to be delimiting the scope of the invention which is defined by the claims.

Example

Lentivirus

The lentiviral backbone which can be used in these methods is based on a self-activating vector described previously (H. Miyoshi et al . , L.Virol. 72, 8750, (1998).

Plasmid pFUGW is based on the HRCS-G vector gift of I. Verma, Salk Insitute, La Jolla, CA) constructed by inserting into its multicloning site the HIV-1 flap sequence polymerase chain reaction (PCR) -amplified from the HIV NLAA3 genome, the human polyubiquitin promoter C (gift of L. Thiel , Amgen, Thousand Oaks, CA) , the* GFP gene and the WRE (gift of D. Trono, University of Geneva, Geneva, Switzerland) . Lentiviral vectors were produced by co-transfecting the transfer vector of pfUGW, the HIV-1 packaging vector Δ8.9 and the VSVG envelope glycoprotein into 293 fibroblasts and concentrated as described previously, FUGW viruses were titered on 293 fibroblasts. Serial dilutions of the virus were applied to the cells, and infectivity was determined after 72 hours by fluorescence microscopy for GPF expression.

The vector was engineered to carry an internal promoter driving the GFP reporter gene. After testing several promoters, the human ubiquitin-C promoter was found to provide the most reliable expression across different cell types and was selected for subsequent experiments . To increase the level of transcription, the wood-chuck hepatitis virus posttranscriptional regulatory element (WRE) was inserted downstream of GFP. To increase the titer of the virus, the human immunodeficiency virus-I (HIV-1) flap element (V. Zennon et al . , Cell 101, 173, (2000)) was inserted between the 5 ' long terminal repeat (LTR) and the human ubiquitin-C internal promoter to generate the viral vector called FUGW. Viruses were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG) and concentrated by ultra-centrifugation to approximately 1x10^s infectious units (I.U.)/μl. The disclosure of the above citations, as far as it concerns the specific methods described herein, shall be incorporated herein by reference.

Mouse oocytes

Female mice (C57Bh/6, 6D2F1 ; IFFA, Credo, France or NMRI Charles River France) aged 5 to 8 weeks were stimulated (day 1) with one peritoneal injection of follicle stimulating hormone (FSH, 5-10u, Folligon; Intervet AG. , Pfaffikon, Switzerland) , followed on day 3 by a second injection (10 IU/0.2 ml) of human chorionic gonadotrophin (HCG, Pregnyl ; Organon, Zurich, Switzerland) to induce ovulation. Females were then mated with normal males from the same strain. The day after the females were killed by cervical dislocation 13 h after HCG administration. The swollen ampullae of the oviducts were dissected; the available oocyte-cumulus complexes were isolated under a stereo microscope in M2 medium containing hyaluronidase (300μg/ml) and maintained under standard incubation conditions (10% C0₂) .

The thus prepared oocytes can then be submitted to the zona pellucida laser-drilling procedure. In the following, said procedure is described in detail:

The set-up used for zona pellucida microdrilling has been described in detail elsewhere (Rink et al . , 1994 Supra; Germond et al . , 1995 Supra) . The disclosure thereof, pertaining to the specific set-up used is explicitly incorporated herein. However, an OCTAX Laser Shot™ device manufactured by OCTAX Microscience GmbH, Germany as described above were focused in the image plane of the microscope by an appropriate microscope objective (magnification 25X) . The power in focus routinely available with the laser was 150 mW.

Using the X-Y microscope stage, each oocyte was positioned to bring a region of the zona pellucida on the aiming spot and the zona pellucida was exposed once or twice to 1-2 ms laser light . Depending on the irradiation time and the temperature of the suspending medium, the diameter of the drilled holes varied between 2-20 μm.

The appropriately prepared and drilled oocytes are brought into contact with the lentiviral vectors. This can be done by incubating the drilled fertilised oocytes with 3 to 5 , preferably 4 hours with lentiviruses carrying a particular transgene .

The transgene can be any transgene of interest, specifically those which will introduce a desired property into a host.

After incubation which is preferably carried out at 20 to 37°C, more preferred at 37°C, under standard incubation conditions (10% C0₂) the embryo can be transferred back to foster mothers the same day.

The results demonstrate that the rate of transgenic production using microdrilling is about the same as that obtained by virus injection, and therefore this method results in a significant advance because of its simplicity and quickness. Specifically, it is to be noted that no micromanipulation of the zygotes is necessary. This provides for the possibility to effectively drill (and thereby introduce transgenes into) a high number of zygotes. As an example, 10-50 zygotes can be placed in one dish at the same time and be drilled by the laser beam within a few minutes. Time-consuming processing of each single zygote is no longer necessary with the procedure of the invention.

Additionally, it could be shown that the inventive concept provides for a very high reproducibility.

Claims

1. Use of a 1.48 μm laser to microdrill a hole in the zona pellucida of a single-cell embryo to introduce a retrovirus carrying a particular transgene into said embryo .

2. Use according to claim 1, characterized in that the hole is between 2-60 μm, , preferably 2-20 μm in diameter.

3. Use according to claim 1 or 2 , characterized in that said lentivirus carrying a particular transgene is incubated with said single-cell embryo for 2-24, preferably 3 to 5, more preferred 4' hours.

4. Use according to any of claims 1-3, characterized in that said single cell embryo is transferred back into a foster mother, on the purpose of obtaining transgenic mice

5. Use according to any of claims 1-4, characterized in that the zona pellucida of said single-cell embryos is exposed to 0.1 μs to 100 ms, preferably 0.1-15 ms of laser light, preferably 0.5 to 10 ms of laser light, more preferably 1 to 5 ms of laser light.

6. Use according to any one of claims 1 - 5, wherein said retrovirus is a lentivirus.