Pulsed electric field transfer of molecules to cells in the body

Abstract

Devices, systems, and methods are provided for delivering molecules, particularly small molecules and / or macromolecules, directly to cells in the body, particularly target cells that will benefit therapeutically from the function of those molecules. In such cases, the molecules are driven into target cells using a pulsed electric field (PEF), which delivers the molecules through the cell wall of the target cell at a desired concentration and at a desired time relative to the delivery of the pulsed electric field, so that the desired gene can exert its desired effect in the target cell. The pulsed electric field energy that drives the molecules into the cell is delivered to the target cell in vivo and in situ. Thus, the molecules are passed into the cell without the use of viruses or ex vivo methods such as in vitro electroporation.

Description

Technical Field

[0001] Cross-Reference to Related Applications

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 061,114 filed on August 4, 2020 entitled "Enhanced Transfer with Pulsed Electric Fields", U.S. Provisional Patent Application No. 63 / 061,091 filed on August 4, 2020 entitled "Pulsed Electric Fields in the Eye", and U.S. Provisional Patent Application No. 63 / 209,335 filed on June 10, 2021 entitled "Induced Extravasation by Energy Delivery to Tissue". The disclosures of all the foregoing applications are hereby incorporated by reference in their entireties.

Background Art

[0002]

[0002] Genes constitute a nucleotide-based storage system for instructions essential for cell survival, function and replication. Each gene is composed of a unique sequence of nucleotides (small segments of DNA) that encode a specific protein with a specific function in an organism. An enzyme transcribes a segment of DNA by constructing a single-stranded molecule of RNA. RNA, like DNA, is a chain of nucleotides. This transcribed RNA (i.e., messenger RNA or mRNA) leaves the nucleus of the cell and travels to the cytoplasm, where ribosomes translate the mRNA to produce the protein specified by the DNA. These proteins are the fundamental building blocks of life.

[0003]

[0003] However, the DNA replication process is not perfect, and mutations occur during replication. In many cases, these mutations are insignificant or may improve or reduce the effectiveness of protein formation. However, sometimes, gene mutations can interfere with the proper functioning of proteins. This can lead to cancer or hereditary disorders, such as many diseases including hemophilia and cystic fibrosis. These traits can be inherited when the gene mutation is hardcoded on the parents' sex chromosomes. In some cases, such as sickle cell anemia, both parents must carry the damaged gene for a disease to develop. Damage to a gene on a single chromosome, such as the conversion from a proto-oncogene to an oncogene that leads to uncontrolled cell division that can result in cancer formation, can also cause disease.

[0004]

[0004] In recent decades, several gene therapies have been developed as attempts to correct or improve the production of proteins affected by such mutations. In some cases, gene therapy involves transplanting the correct version of a gene so that the correct protein is produced. For example, in patients with hemophilia, a gene encoding a coagulation factor is introduced. In other cases, gene therapy induces the upregulation of the expression of specific molecules that enhance the cellular output of the correct protein (for example, inducing cells to produce immunostimulatory molecules to treat diseases such as cancer).

[0005]

[0005] However, introducing genetic material into cells presents challenges. Unlike many other biological molecules and therapeutic compounds, DNA sequences are not designed to cross the cell membrane. Furthermore, DNA sequences lack endocytosis factors that can induce alternative mechanisms for transport into cells. Therefore, all gene therapies require a combination of at least two features: the encoded therapeutic molecule (DNA sequence, gene, siRNA, etc.) and a route of administration to targeted cells. Several approaches have been developed to achieve this.

[0006] Viral vector

[0006] Viruses, including lentiviruses and adeno-associated viruses, are suitable for the delivery of genetic material. Due to their nature, viruses rely on delivering viral DNA or viral RNA to host cells in order to replicate their viral material, and are therefore very promising candidates for gene therapy. The delivery of genes or other genetic material by a vector is called transduction, and infected cells are described as transduced.

[0007]

[0007] When a virus is used for transduction, the disease-causing gene within the virus is removed and replaced with the desired gene that encodes the desired effect (for example, insulin production in the case of a diabetic patient). This is achieved by leaving the gene that allows the virus to infect the host intact. The desired gene is introduced into the virus using a plasmid. A plasmid is a small extrachromosomal DNA molecule inside a cell that is physically separated from chromosomal DNA. Plasmids are most commonly found as small circular double-stranded DNA molecules in bacteria, but in genetic engineering, artificially constructed plasmids are used as vectors. The gene of interest is usually inserted into the vector by various cloning methods such as restriction enzyme ligation, ligation-independent cloning, gateway cloning, and Gibson assembly. The cloning method selected depends on the plasmid. After the cloning process, a reconstructed vector containing the gene of interest is produced.

[0008]

[0008] In some cases, the plasmid itself may not replicate, and the efficacy of the gene effect may diminish over time if the plasmid is degraded, the host cell divides, or the host cell dies. In other cases, the genetic material integrates with the host cell's DNA. Permanently incorporating genetic material into the host's DNA using a viral vector has a more potent effect, but it also carries the risk of introducing both known and unknown risks, and if there is a problem with this gene, it cannot be reversed. Inactivating the disease-inducing aspects of the virus is costly and can be incomplete, and it poses an infection risk to gene therapy patients. The virus may also travel beyond the targeted site of the gene therapy. Viruses can typically infect two or more types of cells. Therefore, when a gene is delivered into a cancer patient's body using a viral vector, the viral vector may infect not only cancer cells but also healthy cells. Similarly, there is a possibility that the new gene may be inserted into the wrong location in the DNA during integration, potentially causing harmful mutations or even cancer in that DNA. This has occurred in clinical trials involving patients with X-linked severe combined immunodeficiency (X-SCID), where transduction of corrective genes into hematopoietic stem cells using retroviruses resulted in T-cell leukemia in 4 out of 20 patients. Furthermore, intentionally introducing pathogenic or non-pathogenic viruses activates the host's immune system. The immune system can reduce the effectiveness of viral gene therapy by interfering with transduction. Moreover, this induction can cause serious side effects in patients, and several deaths associated with virus-based gene therapy have been reported. Ultimately, virus-based gene therapy is costly, risky, and its effectiveness varies considerably, especially with targeted gene therapy.

[0009] Electroporation-mediated gene transfer

[0009] Electroporation has been known for over 30 years as a method for disrupting the integrity of the cell membrane and allowing cells in suspension to take up macromolecules. Typically, electroporation is achieved using an electroporation cuvette 10 and an electroporator 12 as illustrated in Figures 1A to 1B. Referring to Figure 1A, the cuvette 10 is made of glass or plastic and includes a reservoir 14 for receiving a suspension of cells C. A pair of electrode plates 16a, 16b are positioned on the opposite side of the reservoir 14 at a predetermined distance apart. The cells C are suspended in a culture medium transferred to the cuvette 10 by pipette. The cuvette 10 is then placed on the electroporator 12, as illustrated in Figure 1B. The electroporator 12 includes a capacitor that is charged with a high voltage and, when instructed, discharges the stored current in the capacitor toward the sample of cells C in the cuvette 10. The capacitive discharge circuit generates an electrical pulse with a peak voltage and an exponentially decaying waveform. The electric field strength is the voltage applied between electrode plates 16a and 16b, and can be expressed as electric field strength = voltage / distance, where distance is the distance between electrode plates 16A and 16b. The electric field strength and cell size determine the voltage drop across each cell, thus determining the voltage effect in electroporation. Therefore, cell type-specific protocols are usually developed. Similarly, parameters such as field strength, time constant, and decay pulse width can also be changed for each protocol. Thus, the in vitro environment allows for very precise control of transfection conditions, enabling high levels of gene delivery without killing too many cells.

[0010]

[0010] Overall, in vitro transfection is highly reliable for establishing a fundamental understanding of gene action or for creating transgenic experimental animal models. However, providing viable clinical gene therapy remains challenging. When it is necessary to transfect cells in culture medium, several additional steps are required to translate this into patient treatment. In most cases, gene therapy is performed using an apheresis-based approach. This requires sampling of the patient's cells by blood collection or biopsy. The target cell population is then isolated and concentrated. Often, this cell population is amplified to produce transfect cells of a considerable population size. Once the cells are ready, the molecules for gene therapy are electroporated into them. After transfection, the cells are redistributed to the desired site in the patient, such as in the blood or at a target location in the target organ. These steps add substantial burden, cost, invasiveness, time, and risk to the delivery of gene therapy. Overall, these numerous substantial drawbacks prevent pulsed electric field-based gene therapy from being widely adopted as the most promising treatment option.

[0011]

[0011] Overall, gene-based therapies offer a wide range of potential applications, but their high cost, time-consuming nature, risks, and ineffectiveness or inappropriateness for certain disease conditions, along with the limited routes of gene delivery, mean that bridging these therapies to clinical practice is still in its early stages. Therefore, improvements in gene therapy are desired. Such procedures should be safe, effective, and lead to reduced complications. Such procedures should also be applicable to therapies that require the delivery of other types of molecules, particularly macromolecules, into cells. At least some of these objectives will be achieved by the systems, devices, and methods described herein. [Overview of the project]

[0012]

[0012] Embodiments of apparatus, systems and methods for treating target tissues within the body are described herein. Similarly, the present invention relates to the following numbered clauses.

[0013]

[0013] 1. A system for transferring molecules to target tissue cells within a patient's body, An energy delivery device having at least one energy delivery body configured to be positioned near the target tissue cells within the body, A generator electrically connected to the at least one energy delivery body, the generator comprising at least one energy delivery algorithm configured to provide an electrical signal deliverable to the at least one energy delivery body for transmitting pulsed electric field energy that causes at least one of the molecules to enter at least one of the target tissue cells, A system equipped with these features.

[0014]

[0014] 2. The system according to claim 1, wherein the electrical signal comprises a series of pulses, the series of pulses comprising at least one pulse having a positive amplitude and at least one pulse having a negative amplitude.

[0015]

[0015] 3. The system according to claim 2, wherein the series of pulses together have a balance of charge due to positive amplitude on time and negative amplitude on time.

[0016]

[0016] 4. The system according to any one of claims 2 to 3, wherein the series of pulses together have a balance of charge, based on positive amplitude on time and negative amplitude on time, that is sufficient to avoid muscle stimulation within the body.

[0017]

[0017] 5. The system according to any one of claims 2 to 4, wherein the series of pulses together have a charge balance sufficient to avoid ablation of the target tissue cells, due to the positive amplitude on-time and negative amplitude on-time.

[0018]

[0018] 6. The system according to any one of claims 1 to 5, wherein said electrical signal comprises a series of pulses including at least one pulse having different amplitudes or pulse widths.

[0019]

[0019] 7. The system according to any one of claims 1 to 6, wherein said electrical signal comprises a series of pulses, and at least one pulse has a voltage within the range of 10 to 500 V.

[0020]

[0020] 8. The system according to any one of claims 1 to 7, wherein said electrical signal comprises a series of pulses, and at least one pulse has a pulse duration within the range of 0.5 to 200 ms.

[0021]

[0021] 9. The system according to any one of claims 1 to 8, wherein said electrical signal comprises a series of pulses having at least one inter-pulse delay within the range of 10 ms to 10 s.

[0022]

[0022] 10. The system according to any one of claims 1 to 9, wherein said electrical signal comprises a series of pulses having 1 to 100 pulses.

[0023]

[0023] 11. The system according to any one of claims 1 to 10, wherein said electrical signal comprises a series of pulses collectively having a duration of 0.5 to 500 ms.

[0024]

[0024] 12. The system according to claim 1, wherein said electrical signal comprises a series of pulses, at least one pulse has a base pulse width long enough to induce muscle stimulation in said body, and said at least one pulse comprises a plurality of divided pulses having an interval delay sufficient to at least reduce said muscle stimulation.

[0025]

[0025] 13. The system according to claim 12, wherein said base pulse width is within the range of 0.01 to 50,000 μs.

[0026]

[0026] 14. The system according to claim 13, wherein due to the fact that the basic pulse width includes a plurality of section pulses and section delays, the at least one pulse has a pulse width of 1 to 100,000 μs.

[0027]

[0027] 15. The system according to claim 14, wherein the plurality of section pulses includes a maximum of 10,000 section pulses.

[0028]

[0028] 16. The system according to claim 15, wherein each section pulse has a duration in the range of 0.05 to 5 μs.

[0029]

[0029] 17. The system according to claim 15, wherein each section delay has a duration in the range of 0.001 to 10 μs.

[0030]

[0030] 18. The system according to claim 14, wherein each section pulse has a duration in the range of 0.05 to 50 μs.

[0031]

[0031] 19. The system according to claim 14, wherein each section delay has a duration in the range of 0.001 to 100 ms.

[0032]

[0032] 20. The system according to claim 14, wherein the at least one section pulse has an on-time not exceeding 10 μs.

[0033]

[0033] 21. The system according to claim 12, wherein the at least one pulse includes at least two pulses separated by a delay of 10 ms to 10 s.

[0034]

[0034] 22. The system according to claim 21, wherein the at least two pulses have opposite polarities.

[0035]

[0035] 23. The system according to claim 22, wherein each of the pulses in the series of pulses has a basic pulse width of sufficient length to cause muscle stimulation in the body, and each of the pulses in the series of pulses comprises a plurality of interval pulses having an interval delay sufficient to at least reduce the muscle stimulation.

[0036]

[0036] 24. The system according to claim 23, wherein the pulses in the series of pulses together have a balance of charge due to positive amplitude on time and negative amplitude on time.

[0037]

[0037] 25. The system according to any one of claims 12 to 24, wherein the at least one pulse has a voltage in the range of 10 to 250 V.

[0038]

[0038] 26. The system according to claim 1, wherein the electrical signal comprises a series of biphasic pulses, at least one biphasic pulse having a cycle length of 0.01 to 10 μs, and the at least one biphasic pulse comprises a plurality of interval pulses having interval delays.

[0039]

[0039] 27. The system according to claim 26, wherein the plurality of interval pulses include up to 10,000 interval pulses.

[0040]

[0040] 28. The system according to claim 27, wherein each interval pulse has a duration in the range of 0.004 to 0.4 μs.

[0041]

[0041] 29. The system according to claim 28, wherein each interval delay has a duration in the range of 10 to 10,000 μs.

[0042]

[0042] 30. The system according to claim 28, wherein each interval pulse has a duration in the range of 0.05 to 50 μs.

[0043]

[0043] 31. The system according to claim 26, wherein each interval delay has a duration in the range of 0.001 to 100 ms.

[0044]

[0044] 32. The system according to claim 26, wherein at least one interval pulse has an on time not exceeding 10 μs.

[0045]

[0045] 33. The system according to claim 26, wherein the series of biphasic pulses comprises up to 1000 cycles.

[0046]

[0046] 34. The system according to any one of claims 26 to 33, wherein the series of biphasic pulses are grouped into packets having inter-packet delays.

[0047]

[0047] 35. The system according to any one of claims 26 to 34, wherein the at least one biphase pulse has a voltage in the range of 500 to 2000 V.

[0048]

[0048] 36. The system according to claim 1, wherein the electrical signal comprises a series of pulses, the series of pulses comprising at least one high-voltage high-frequency pulse followed by at least one low-voltage low-frequency pulse, the high voltage being in the range of 100 to 1000 V, the high frequency having a pulse width of 50 ns to 1 ms, the low voltage being in the range of 5 to 100 V, and the low frequency having a pulse width of 1 ms to 50 ms.

[0049]

[0049] 37. The system according to claim 36, wherein the at least one low voltage low frequency pulse comprises at least two low voltage low frequency pulses having opposite polarities.

[0050]

[0050] 38. The system according to claim 36, wherein the at least one low-voltage low-frequency pulse includes a DC waveform.

[0051]

[0051] 39. The system according to claim 36, wherein the at least one high-voltage high-frequency pulse includes a biphasic waveform.

[0052]

[0052] 40. The system according to claim 36, wherein the at least one high-voltage high-frequency pulse is separated from the at least one low-voltage low-frequency pulse by a delay of 100 μs to 2 seconds.

[0053]

[0053] 41. The system according to any one of claims 1 to 40, wherein the electrical signal comprises a series of pulses, and at least one pulse comprises at least one spike.

[0054]

[0054] 42. The system according to claim 41, wherein the at least one pulse has a primary voltage, and the at least one spike includes a plurality of spikes formed by oscillation near the primary voltage.

[0055]

[0055] 43. The system according to claim 42, wherein the oscillation is within the range of 1 to 25% of the primary voltage.

[0056]

[0056] 44. The system according to claim 43, wherein the oscillation is within the range of 1 to 10% of the primary voltage.

[0057]

[0057] 45. The system according to any one of claims 41 to 44, wherein the at least one pulse includes a plurality of spikes that are not evenly distributed along the at least one pulse.

[0058]

[0058] 46. The system according to any one of claims 41 to 45, wherein the series of pulses is biphasic.

[0059]

[0059] 47. The system according to any one of claims 1 to 46, wherein the energy delivery device comprises a shaft having one or more tines from which it can be extended.

[0060]

[0060] 48. The system according to claim 47, wherein the energy delivery device is configured to deliver the molecule through at least one of the one or more tines.

[0061]

[0061] 49. The system according to claim 48, wherein the energy delivery device is configured to deliver a molecule different from at least one of the one or more tines through at least one of the one or more tines from at least one other tine of the one or more tines.

[0062]

[0062] 50. The system according to any one of claims 47 to 49, wherein the at least one energy delivery body comprises at least one of the one or more tines.

[0063]

[0063] 51. The system according to claim 50, wherein the at least one energy delivery body comprises at least two of the one or more tines that can be individually energized.

[0064]

[0064] 52. The system according to any one of claims 1 to 51, wherein the at least one energy delivery body comprises a cage electrode.

[0065]

[0065] 53. The system according to any one of claims 1 to 52, wherein the energy delivery device comprises an elongated shaft, and the at least one energy delivery body comprises at least two projections, each projection extending radially outward from the elongated shaft.

[0066]

[0066] 54. The system according to any one of claims 1 to 53, wherein the generator comprises at least one other energy delivery algorithm configured to provide another electrical signal deliverable to the at least one energy delivery body in order to deliver conditioning energy that induces extravasation of fluid from the body within a local region of the target tissue cells.

[0067]

[0067] 55. The system according to claim 54, wherein the extravasation is from the vascular structure of the patient's body.

[0068]

[0068] 56. A system such as any one of claims 54 to 55, wherein the local region includes the interstitial space around the target tissue cells.

[0069]

[0069] 57. The system according to any one of claims 54 to 56, wherein the molecule is delivered to the vascular structure of the body, and the induced extravasation delivers the molecule from the vascular structure to the local area.

[0070]

[0070] 58. The system according to any one of claims 54 to 57, wherein the other electrical signal comprises a plurality of monophase pulses, each pulse having a duration of more than 500 microseconds.

[0071]

[0071] 59. The system according to any one of claims 54 to 57, wherein the other electrical signal comprises a plurality of pulses, at least one of the plurality of pulses having a positive amplitude and at least one of the plurality of pulses having a negative amplitude.

[0072]

[0072] 60. The system according to claim 59, wherein each of the plurality of pulses has a duration of more than 500 microseconds.

[0073]

[0073] 61. The system according to any one of claims 54 to 60, wherein the energy delivery device comprises at least one pressure sensor.

[0074]

[0074] 62. The system according to claim 61, wherein the at least one pressure sensor is configured to monitor the effects of extravasation and to provide sensor feedback data.

[0075]

[0075] 63. The system according to claim 62, wherein the system comprises a mechanism for providing the user with feedback data of the sensor or information based on the feedback data of the sensor.

[0076]

[0076] 64. The system according to claim 62, wherein the generator comprises a processor configured to modify the at least one energy delivery algorithm or switch to a different energy delivery algorithm based on feedback data from the sensor in order to deliver energy that modulates the induction of extravasation.

[0077]

[0077] 65. The system according to any one of claims 1 to 60, wherein the system comprises at least one sensor.

[0078]

[0078] 66. The system according to claim 65, wherein the at least one sensor includes a sensor that monitors pressure, temperature, impedance, resistance, capacitance, conductivity, pH, optical properties, coherence, echo brightness, fluorescence, dielectric constant, light permittivity and / or conductance.

[0079]

[0079] 67. The system according to any one of claims 1 to 66, wherein the generator comprises an additional energy delivery algorithm configured to provide a deliverable ablation electrical signal to the at least one energy delivery body in order to deliver ablation energy causing at least one of the target tissue cells to die.

[0080]

[0080] 68. The system according to any one of claims 1 to 67, wherein the at least one molecule comprises a small molecule and / or a polymer.

[0081]

[0081] 69. The system according to any one of claims 1 to 68, wherein the at least one molecule comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomeric molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolytically targeted chimera, a zinc finger nuclease or a transcription activator-like effector nuclease, a protein and / or material that induces a genetic or epigenetic change in cellular behavior.

[0082]

[0082] 70. The system according to any one of claims 1 to 69, wherein the at least one molecule comprises at least one gene greater than 5 kb.

[0083]

[0083] 71. The system according to claim 70, wherein the at least one molecule comprises at least one gene greater than 10 kb.

[0084]

[0084] 72. The system according to any one of claims 1 to 71, wherein the target tissue cells include retinal cells.

[0085]

[0085] 73. The system according to claim 72, wherein the retinal cells include cells of the cone layer and / or cells of the retinal pigment epithelium.

[0086]

[0086] 74. The system according to any one of claims 1 to 71, wherein the target tissue cells include cells of bone marrow.

[0087]

[0087] 75. The system according to any one of claims 1 to 71, wherein the target tissue cells include cells of the digestive system, including the liver, pancreas, intestine and / or colon.

[0088]

[0088] 76. The system according to any one of claims 1 to 71, wherein the target tissue cells include heart cells.

[0089]

[0089] 77. A system for treating target tissue cells within a patient's body, wherein the system is An energy delivery device comprising a shaft having one or more tines extending therefrom, wherein the one or more tines comprise a first energy delivery body and a second energy delivery body, and the one or more tines are configured to be positioned near the target tissue cells in the patient's body, and A generator electrically connected to the first and second energy carriers, wherein the generator comprises at least a first energy delivery algorithm configured to provide a first electrical signal deliverable to the first energy carrier and at least a second energy delivery algorithm configured to provide a second electrical signal deliverable to the second energy carrier. A system equipped with these features.

[0090]

[0090] 78. The system according to claim 77, wherein the first electrical signal generates energy to transfer at least one molecule to at least one of the target tissue cells.

[0091]

[0091] 79. The system according to any one of claims 77 to 78, wherein the second electrical signal generates energy that causes ablation of at least one target tissue cell.

[0092]

[0092] 80. The system according to any one of claims 77 to 79, wherein at least one of the one or more tines extends distally from the distal end of the shaft along the longitudinal axis of the shaft, and the second energy deliverer is positioned along it.

[0093]

[0093] 81. The system according to any one of claims 77 to 80, wherein at least one of the one or more tines extends radially from the shaft and the first energy deliverer is positioned along it.

[0094]

[0094] 82. The system according to claim 77, wherein one or more tines are configured to enable ablation of a first region of the target tissue cell and the transfer of molecules to a second region of the target tissue cell.

[0095]

[0095] 83. The system according to claim 82, wherein the second region at least partially surrounds the first region.

[0096]

[0096] 84. The system according to any one of claims 77 to 83, wherein the energy delivery device is configured such that at least one of the one or more tines can deliver a plurality of molecules.

[0097]

[0097] 85. The system according to claim 84, wherein the energy delivery device is configured to deliver a molecule different from at least one of the one or more tines through at least one of the one or more tines from at least one other tine of the one or more tines.

[0098]

[0098] 86. The system according to any one of claims 77 to 85, wherein at least one of the one or more tines is adjustable and extendable.

[0099]

[0099] 87. A method for transferring molecules to cells of a target tissue in a patient's body, A step of delivering multiple molecules to the patient's body, A step of positioning at least one energy delivery body of an energy delivery device within a range of target tissue sufficient to receive pulsed electric field energy delivered therefrom, and A step of delivering the pulsed electric field energy to the at least one energy deliverer in such a way that at least one of the molecules enters at least one of the cells of the target tissue. Methods that include...

[0100]

[0100] 88. The method according to claim 87, wherein the target tissue cells directly receive therapeutic benefits from the function of the molecule.

[0101]

[0101] 89. The method of claim 88, wherein the direct therapeutic benefit includes the treatment of a disorder.

[0102]

[0102] 90. The method according to claim 89, wherein the disorder includes a hereditary disorder.

[0103]

[0103] 91. The method according to any one of claims 87 to 90, wherein the at least one molecule comprises a small molecule and / or a polymer.

[0104]

[0104] 92. The method according to any one of claims 87 to 91, wherein the at least one molecule comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomeric molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolytically targeted chimera, a zinc finger nuclease or a transcription activator-like effector nuclease, a protein and / or material that induces a genetic or epigenetic change in cellular behavior.

[0105]

[0105] 93. The method according to any one of claims 87 to 92, wherein the at least one molecule comprises at least one gene greater than 5 kb.

[0106]

[0106] 94. The method according to claim 93, wherein the at least one molecule comprises at least one gene greater than 10 kb.

[0107]

[0107] 95. The method according to any one of claims 87 to 94, wherein the step of delivering the plurality of molecules to the body includes delivering the molecules into the veins of the body.

[0108]

[0108] 96. The method according to claims 87 to 94, wherein the step of delivering the plurality of molecules to the body includes delivering the molecules into the veins of the body and locally to the target tissue.

[0109]

[0109] 97. The method according to any one of claims 87 to 96, wherein the step of delivering the plurality of molecules is performed at least before delivering the energy.

[0110]

[0110] 98. The method according to any one of claims 87 to 97, wherein the step of delivering the plurality of molecules includes delivering the plurality of molecules to a plurality of locations in or near the target tissue.

[0111]

[0111] 99. The method according to claim 98, wherein the plurality of locations are within 0.5 mm to 5 cm of at least one cell of the target tissue.

[0112]

[0112] 100. The method according to any one of claims 98 to 99, wherein delivering the plurality of molecules to the plurality of locations includes delivering molecular solutions of various concentrations, molecular solutions of various volumes, and / or molecular solutions of various types to one or more of the plurality of locations.

[0113]

[0113] 101. The method according to any one of claims 87 to 99, wherein delivering the plurality of molecules to a plurality of locations includes delivering a molecule to at least one of the plurality of locations that is different from the molecule at another of the plurality of locations.

[0114]

[0114] 102. The method according to any one of claims 87 to 101, wherein the energy delivery device comprises a shaft having one or more tines from which it can be extended, and the delivery of the plurality of molecules to a plurality of locations is achieved by delivering the molecules through one or more of the one or more tines.

[0115]

[0115] 103. The method according to claim 102, wherein delivering the plurality of molecules includes delivering molecules different from at least one of the one or more tines through at least one of the one or more tines.

[0116]

[0116] 104. The method according to claim 102, wherein the energy delivery device comprises a shaft having one or more tines extendable therefrom, the at least one energy delivery body comprises at least one of the one or more tines, and the delivery of the energy comprises energizing at least one of the plurality of tines.

[0117]

[0117] 105. The method according to claim 104, wherein energizing at least one of the plurality of tines includes energizing at least one of the plurality of tines individually without energizing at least one of the plurality of tines.

[0118]

[0118] 106. The method according to any one of claims 87 to 105, further comprising the step of inducing extravasation of fluid within a local area of ​​the body.

[0119]

[0119] 107. The method according to claim 106, wherein the step of inducing extravasation is performed before energy delivery.

[0120]

[0120] 108. The method according to any one of claims 106 to 107, wherein the extravasation increases the delivery of molecules to the target tissue.

[0121]

[0121] 109. The method according to any one of claims 106 to 108, wherein the step of delivering the plurality of molecules includes delivering the plurality of molecules to the vascular structure of the body, and the extravasation is from the vascular structure.

[0122]

[0122] 110. The method according to claim 106, wherein the step of inducing extravasation comprises delivering conditioning energy to the at least one energy delivery body in order to transfer conditioning energy to the body in a manner that induces extravasation of fluid from the body within a local area of ​​the target tissue.

[0123]

[0123] 111. The method according to claim 110, wherein the conditioning energy is from another electrical signal comprising a plurality of monophase pulses, each pulse having a duration of more than 500 microseconds.

[0124]

[0124] 112. The method according to any one of claims 110 to 111, wherein the conditioning energy includes PEF energy.

[0125]

[0125] 113. The method according to claim 87, further comprising the step of delivering conditioning energy to the target tissue before delivering the pulsed electric field energy.

[0126]

[0126] 114. The method according to claim 113, wherein the conditioning increases the cellular resistance of the target tissue to eventual cell death.

[0127]

[0127] 115. The method according to any one of claims 87 to 114, further comprising the step of preheating the target tissue before delivering the energy.

[0128]

[0128] 116. The method according to claim 115, wherein the step of preheating the target tissue includes delivering conditioning energy to the target tissue.

[0129]

[0129] 117. The method according to any one of claims 87 to 116, wherein the pulsed electric field energy is from an electrical signal comprising a series of pulses, the series of pulses comprising at least one pulse having a positive amplitude and at least one pulse having a negative amplitude.

[0130]

[0130] 118. The method according to claim 117, wherein the series of pulses together have a balance of charge due to positive amplitude on time and negative amplitude on time.

[0131]

[0131] 119. The method according to claim 117, wherein the series of pulses together have a balance of charge, based on positive amplitude on time and negative amplitude on time, that is sufficient to avoid muscle stimulation within the body.

[0132]

[0132] 120. The method according to claim 117, wherein the series of pulses together have a charge balance of positive amplitude on time and negative amplitude on time that is sufficient to avoid ablation of the target tissue cells.

[0133]

[0133] 121. A system for treating a portion of a patient's eye, wherein the system is An instrument having at least one electrode body configured to be positioned in, above, or near the eye, and A generator electrically connected to the at least one electrode body, the generator comprising at least one energy delivery algorithm configured to provide an electrical signal of pulsed electric field energy deliverable to the at least one electrode body in order to cause at least one molecule to enter the eye cells, A system equipped with these features.

[0134]

[0134] 122. The system according to claim 121, wherein the instrument comprises a shaft having a distal end, and the at least one electrode body is positioned near the distal end of the shaft.

[0135]

[0135] 123. The system according to any one of claims 121 to 122, wherein the shaft is configured to be insertable into the vitreous cavity of the eye.

[0136]

[0136] 124. The system according to any one of claims 121 to 123, wherein at least one electrode body is configured to be insertable into a subretinal bleb inside the eye.

[0137]

[0137] 125. The system according to any one of claims 121 to 123, wherein at least one electrode body is configured to be insertable into the suprachoroidal space inside the eye.

[0138]

[0138] 126. The system according to any one of claims 121 to 123, wherein the at least one electrode body includes a bipolar pair of electrode bodies.

[0139]

[0139] 127. The system according to claim 126, wherein the bipolar pair of electrodes includes a first electrode positioned to be positioned within a subretinal bleb, and a second electrode positioned to be positioned outside the subretinal bleb.

[0140]

[0140] 128. The system according to claim 126, wherein the bipolar pair of electrodes includes a first electrode body positioned to be positioned within the suprachoroidal lumen, and a second electrode body positioned to be positioned outside the suprachoroidal lumen.

[0141]

[0141] 129. The system according to claim 126, wherein the bipolar electrode body includes a first electrode body positioned to be positioned within the vitreous cavity, and a second electrode body positioned to be positioned outside the vitreous cavity.

[0142]

[0142] 130. The system according to claim 126, wherein the bipolar electrode body includes a first electrode body positioned to be locatable at a first position, and a second electrode body positioned to be locatable at a second position, and a portion of the retina is positioned between the first position and the second position.

[0143]

[0143] 131. The system according to claim 126, wherein the polarity of the bipolar pair of electrodes is reversible.

[0144]

[0144] 132. The system according to any one of claims 121 to 125, further comprising a return electrode configured to be positioned at a certain distance from at least one of the at least one electrode bodies so that the at least one electrode body functions in a unipolar form.

[0145]

[0145] 133. The system according to claim 132, wherein the return electrode is configured to be positioned in contact with or near the outer surface of the eye.

[0146]

[0146] 134. The system according to claim 133, wherein the return electrode includes an electroretinography electrode, a microscope, a contact lens, or a tweezertrode electrode.

[0147]

[0147] 135. The system according to claim 132, wherein the return electrode is configured to be positioned at least partially in the posterior ocular cavity.

[0148]

[0148] 136. The system according to claim 132, wherein the return electrode is configured to be positioned at least partially in the choroidal space.

[0149]

[0149] 137. The system according to any one of claims 121 to 136, wherein the instrument comprises a lumen for delivering fluid.

[0150]

[0150] 138. The system according to claim 137, wherein the instrument comprises at least one outlet in fluid communication with the lumen such that the fluid can be delivered near at least one of the electrode bodies.

[0151]

[0151] 139. The system according to claim 137, further comprising the fluid, wherein the fluid comprises small molecules and / or polymers.

[0152]

[0152] 140. The system according to any one of claims 121 to 139, wherein the at least one molecule comprises a small molecule and / or a polymer.

[0153]

[0153] 141. The system according to any one of claims 121 to 139, wherein the at least one molecule comprises a plasmid, DNA, a synthetic DNA vector, RNA, a nucleic acid-based molecule, an antisense oligonucleotide, an oligomeric molecule, a ribozyme, a ribonucleoprotein, CRISPR, a recombinant protein, a proteolytically targeted chimera, a zinc finger nuclease or a transcription activator-like effector nuclease, a protein and / or material that induces a genetic or epigenetic change in cellular behavior.

[0154]

[0154] 142. The system according to any one of claims 121 to 141, wherein the at least one molecule comprises at least one gene greater than 5 kb.

[0155]

[0155] 143. The system according to claim 142, wherein the at least one molecule comprises at least one gene greater than 10 kb.

[0156]

[0156] 144. The system according to any one of claims 121 to 143, wherein the cells are located within the retina of the eye.

[0157]

[0157] 145. The system according to claim 144, wherein the cells are arranged within the rod-cone layer of the retina.

[0158]

[0158] 146. The system according to claim 144, wherein the cells are located within the retinal pigment epithelium of the retina.

[0159]

[0159] 147. A method for treating a patient's eye, A step of positioning at least one electrode body inside or near the eye, A step of introducing at least one molecule into a portion of the eye, and The process of delivering pulsed electric field energy to the at least one electrode body in order to cause the at least one molecule to enter the cells inside the eye. Methods that include...

[0160]

[0160] 148. The method according to claim 147, wherein the pulsed electric field energy comprises a series of monophase pulses.

[0161]

[0161] 149. The method according to claim 148, wherein the series of monophase pulses includes a series of low-voltage pulses.

[0162]

[0162] 150. The method according to claim 147, wherein the pulsed electric field energy comprises a series of biphase pulses.

[0163]

[0163] 151. The method according to any one of claims 147 to 150, wherein the step of positioning the at least one electrode body includes positioning the at least one electrode body within the vitreous cavity of the eye.

[0164]

[0164] 152. The method according to any one of claims 147 to 150, wherein the step of positioning the at least one electrode body includes positioning the at least one electrode body within a subretinal bleb inside the eye.

[0165]

[0165] 153. The method according to any one of claims 147 to 150, wherein the step of positioning the at least one electrode body includes positioning the at least one electrode body in the suprachoroidal space inside the eye.

[0166]

[0166] 154. The method according to claim 147, wherein the at least one electrode body includes a bipolar pair of electrode bodies, and the step of positioning the at least one electrode body includes positioning a first electrode body within a subretinal bleb and positioning a second electrode body outside the subretinal bleb.

[0167]

[0167] 155. The method according to claim 147, wherein the at least one electrode body includes a bipolar pair of electrode bodies, and the step of positioning the at least one electrode body includes positioning the first electrode body in the suprachoroidal space and positioning the second electrode body outside the suprachoroidal space.

[0168]

[0168] 156. The method according to claim 147, wherein the at least one electrode body includes a bipolar pair of electrode bodies, and the step of positioning the at least one electrode body includes positioning a first electrode body inside the vitreous cavity and positioning a second electrode body outside the vitreous cavity.

[0169]

[0169] 157. The method according to claim 147, wherein the at least one electrode body includes a bipolar pair of electrode bodies, and the step of positioning the at least one electrode body includes positioning the first electrode body in a first position and the second electrode body in a second position, and a portion of the retina is positioned between the first position and the second position.

[0170]

[0170] 158. The method according to any one of claims 154 to 157, further comprising the step of reversing the polarity of the bipolar pair.

[0171]

[0171] 159. The method according to claim 147, further comprising the step of delivering energy to the at least one electrode body such that the cells are located inside the layers of the retina of the eye and the at least one molecule enters cells inside different layers of the retina.

[0172]

[0172] 160. The method according to claim 147, further comprising the step of positioning a return electrode at a certain distance from the at least one electrode body so that the at least one electrode body can function in a unipolar form.

[0173]

[0173] 161. The method according to claim 160, wherein the step of positioning the return electrode includes positioning the return electrode in contact with or near the outer surface of the eye.

[0174]

[0174] 162. The method according to claim 161, wherein the return electrode includes an electroretinography electrode, a microscope, a contact lens, or a tweezerstrode electrode.

[0175]

[0175] 163. The method according to claim 160, wherein the step of positioning the return electrode includes positioning the return electrode at least partially in the posterior ocular cavity.

[0176]

[0176] 164. The method according to claim 160, wherein the step of positioning the return electrode includes positioning the return electrode at least partially in the choroidal space.

[0177]

[0177] 165. The method according to any one of claims 147 to 164, wherein the at least one molecule comprises at least one small molecule and / or polymer.

[0178]

[0178] 166. The method according to any one of claims 147 to 164, wherein the at least one molecule comprises a plasmid, RNA, nucleic acid-based molecule, antisense oligonucleotide, oligomeric molecule, ribozyme, ribonucleoprotein, CRISPR, recombinant protein, protein and / or material that induces genetic or epigenetic changes in cellular behavior.

[0179]

[0179] 167. The method according to any one of claims 147 to 164, wherein the at least one molecule comprises a synthetic DNA vector.

[0180]

[0180] 168. The method according to any one of claims 147 to 167, wherein the cells are located within the retina of the eye.

[0181]

[0181] 169. The method according to claim 168, wherein the cells are arranged within the rod-cone layer of the retina.

[0182]

[0182] 170. The method according to claim 168, wherein the cells are located within the retinal pigment epithelium of the retina.

[0183]

[0183] These and other embodiments will be described in further detail in the following description relating to the attached drawings. The present invention provides, for example, the following items: (Item 1) A system for transferring molecules to target tissue cells within the patient's body, An energy delivery device having at least one energy delivery body configured to be positioned near the target tissue cells within the body, A system comprising: a generator electrically connected to the at least one energy delivery body, the generator comprising at least one energy delivery algorithm configured to provide an electrical signal deliverable to the at least one energy delivery body for transmitting pulsed electric field energy causing at least one of the molecules to enter at least one of the target tissue cells. (Item 2) The aforementioned electrical signal includes a series of pulses, The system according to item 1, wherein the series of pulses includes at least one pulse having a positive amplitude and at least one pulse having a negative amplitude. (Item 3) The system described in item 2, wherein the series of pulses together have a balance of charge due to positive amplitude on-time and negative amplitude on-time. (Item 4) The system according to item 2 or 3, wherein the series of pulses together have a sufficient balance of charge, based on positive amplitude on-time and negative amplitude on-time, to avoid muscle stimulation within the body. (Item 5) The system according to any one of items 2 to 4, wherein the series of pulses together have a sufficient balance of charge, based on positive amplitude on-time and negative amplitude on-time, to avoid ablation of the target tissue cells. (Item 6) The system according to any one of items 1 to 5, wherein the electrical signal comprises a series of pulses, each containing at least one pulse with a different amplitude or pulse width. (Item 7) The aforementioned electrical signal includes a series of pulses, A system as described in any one of items 1 to 6, wherein at least one pulse has a voltage in the range of 10 to 500V. (Item 8) The aforementioned electrical signal includes a series of pulses, A system as described in any one of items 1 to 7, wherein at least one pulse has a pulse duration in the range of 0.5 to 200 ms. (Item 9) The system according to any one of items 1 to 8, wherein the electrical signal includes a series of pulses having at least one inter-pulse delay in the range of 10 ms to 10 s. (Item 10) The aforementioned electrical signal includes a series of pulses, At least one pulse has a basic pulse width of sufficient length to cause muscle stimulation within the body, The system according to item 1, wherein the at least one pulse comprises a plurality of interval pulses having an interval delay sufficient to at least reduce the muscle stimulation. (Item 11) The system according to item 10, wherein the at least one interval pulse has an on-time not exceeding 10 μs. (Item 12) The system according to item 10, wherein the at least one pulse includes at least two pulses separated by a delay of 10 ms to 10 s. (Item 13) The system according to item 12, wherein at least two of the pulses have opposite polarities. (Item 14) Each of the pulses in the series of pulses has a basic pulse width of sufficient length to cause muscle stimulation within the body. The system according to item 13, wherein each of the pulses in the series of pulses comprises a plurality of interval pulses having an interval delay sufficient to at least reduce the muscle stimulation. (Item 15) The system according to item 14, wherein the pulses in the series of pulses together have a charge balance due to positive amplitude on time and negative amplitude on time. (Item 16) The aforementioned electrical signal includes a series of biphase pulses, At least one biphasic pulse has a cycle length of 0.01 to 10 μs. The system according to item 1, wherein the at least one biphasic pulse comprises a plurality of interval pulses having interval delays. (Item 17) The system described in item 16, wherein the series of biphasic pulses are grouped into packets having inter-packet delays. (Item 18) The aforementioned electrical signal includes a series of pulses, The series of pulses includes at least one high-voltage high-frequency pulse followed by at least one low-voltage low-frequency pulse. High voltage is in the range of 100 to 1000V. High frequency has a pulse width of 50 ns to 1 ms. Low voltage is within the range of 5 to 100V. The low-frequency system is the one described in item 1, having a pulse width of 1 ms to 50 ms. (Item 19) The system according to item 18, wherein the at least one low-voltage low-frequency pulse comprises at least two low-voltage low-frequency pulses having opposite polarities. (Item 20) The system described in item 18, wherein the at least one low-voltage, low-frequency pulse includes a DC waveform. (Item 21) The system according to item 18, wherein the at least one high-voltage high-frequency pulse includes a biphasic waveform. (Item 22) The aforementioned electrical signal includes a series of pulses, A system as described in any one of items 1 through 21, wherein at least one pulse includes at least one spike. (Item 23) The at least one pulse has a primary voltage, The system according to item 22, wherein the at least one spike includes a plurality of spikes formed by oscillation near the primary voltage. (Item 24) The energy delivery device comprises a shaft having one or more tines extendable therefrom, according to any one of items 1 to 23. (Item 25) The system according to any one of items 1 to 24, wherein the at least one energy delivery body comprises a cage electrode. (Item 26) The energy delivery device comprises an elongated shaft, The at least one energy delivery body comprises at least two protrusions, Each projection extends radially outward from the elongated shaft, the system as described in any one of items 1 to 25. (Item 27) The system according to any one of items 1 to 26, wherein the generator comprises at least one other energy delivery algorithm configured to provide another electrical signal deliverable to the at least one energy delivery body in order to deliver conditioning energy that induces extravasation of fluid from the body within a local region of the target tissue cells. (Item 28) The local region includes the interstitial space around the target tissue cells, as described in item 27. (Item 29) The molecule is delivered to the vascular structure of the body, The system according to item 27 or 28, wherein the induced extravasation delivers the molecule from the vascular structure to the local region. (Item 30) The aforementioned other electrical signal includes a plurality of monophase pulses, Each pulse has a duration of more than 500 microseconds, as described in any one of items 27 to 29. (Item 31) The aforementioned other electrical signal includes multiple pulses, At least one of the plurality of pulses has a positive amplitude, The system according to any one of items 27 to 29, wherein at least one of the plurality of pulses has a negative amplitude. (Item 32) The system is the system described in any one of items 1 to 31, comprising at least one sensor. (Item 33) The system according to item 32, wherein the at least one sensor includes a sensor that monitors pressure, temperature, impedance, resistance, capacitance, conductivity, pH, optical properties, coherence, echo brightness, fluorescence, dielectric constant, photodielectric constant and / or conductance. (Item 34) The system according to any one of items 1 to 33, wherein the generator comprises an additional energy delivery algorithm configured to provide a deliverable ablation electrical signal to the at least one energy delivery body in order to deliver ablation energy that kills at least one of the target tissue cells. (Item 35) The system according to any one of items 1 to 34, wherein the at least one molecule includes small molecules and / or polymers. (Item 36) The system according to any one of items 1 to 35, wherein the at least one molecule includes a plasmid, DNA, synthetic DNA vector, RNA, nucleic acid-based molecule, antisense oligonucleotide, oligomeric molecule, ribozyme, ribonucleoprotein, CRISPR, recombinant protein, proteolytically targeted chimera, zinc finger nuclease or transcription activator-like effector nuclease, protein and / or material that induces genetic or epigenetic changes in cellular behavior. (Item 37) The system described in any one of items 1 to 36, wherein the at least one molecule comprises at least one gene greater than 5 kb. (Item 38) The system described in item 37, wherein the at least one molecule comprises at least one gene greater than 10 kb. (Item 39) The target tissue cells include retinal cells, as described in any one of items 1 to 38. (Item 40) The target tissue cells include bone marrow cells, as described in any one of items 1 to 38. (Item 41) The target tissue cells include cells of the digestive system, including the liver, pancreas, intestines and / or colon, as described in any one of items 1 to 38. (Item 42) The aforementioned target tissue cells include cardiac cells, as described in any one of items 1 to 38. (Item 43) A system for treating target tissue cells within a patient's body, wherein the system is An energy delivery device comprising a shaft having one or more tines extending therefrom, wherein the one or more tines comprise a first energy delivery body and a second energy delivery body, and the one or more tines are configured to be positioned near the target tissue cells within the patient's body, A system comprising: a generator electrically connected to the first and second energy carriers, the generator comprising at least a first energy delivery algorithm configured to provide a first electrical signal deliverable to the first energy carrier and at least a second energy delivery algorithm configured to provide a second electrical signal deliverable to the second energy carrier. (Item 44) The system according to item 43, wherein the first electrical signal generates energy to transfer at least one molecule to at least one of the target tissue cells. (Item 45) The system according to item 43 or 44, wherein the second electrical signal generates energy that causes ablation of at least one target tissue cell. (Item 46) At least one of the one or more tines extends distally from the distal end of the shaft along the longitudinal axis of the shaft, The second energy delivery body is arranged along with the system described in any one of items 43 to 45. (Item 47) At least one of the one or more tines extends radially from the shaft, The first energy delivery body is arranged along with the system described in any one of items 43 to 46. (Item 48) The system according to item 43, wherein one or more tines are configured to allow ablation of a first region of the target tissue cell and the transfer of molecules to a second region of the target tissue cell. (Item 49) The system described in item 48, wherein the second region encloses the first region at least partially. (Item 50) The energy delivery device is a system according to any one of items 43 to 49, wherein at least one of the one or more tines is configured to deliver multiple molecules. (Item 51) A system for treating a portion of a patient's eye, the system comprising: an instrument having at least one electrode body configured to be positioned inside, on, or near the eye; and a generator electrically connected to the at least one electrode body, the generator comprising at least one energy delivery algorithm configured to provide an electrical signal of pulsed electric field energy deliverable to the at least one electrode body in order to introduce at least one molecule into the cells of the eye. (Item 52) The aforementioned device comprises a shaft having a distal end, The system according to item 51, wherein the at least one electrode body is located near the distal end of the shaft. (Item 53) The system according to item 51 or 52, wherein the shaft is configured to be insertable into the vitreous cavity of the eye. (Item 54) The system according to any one of items 51 to 53, wherein the at least one electrode body is configured to be insertable into a subretinal bleb inside the eye. (Item 55) The system according to any one of items 51 to 54, wherein the at least one electrode body includes a bipolar pair of electrode bodies. (Item 56) The system according to any one of items 51 to 55, further comprising a return electrode configured to be positioned at a certain distance from the at least one electrode so that the at least one electrode functions in a unipolar form. (Item 57) The system according to item 56, wherein the return electrode is configured to be positioned in contact with or near the outer surface of the eye. (Item 58) The system according to item 56, wherein the return electrode is configured to be positioned at least partially in the posterior ocular cavity. (Item 59) The apparatus is a system according to any one of items 51 to 58, comprising a lumen for delivering fluid. (Item 60) The system according to item 59, wherein the apparatus comprises at least one outlet in fluid communication with the lumen so that the fluid can be delivered near at least one of the electrode bodies. (Item 61) The system according to any one of items 51 to 60, wherein the at least one molecule includes small molecules and / or polymers. (Item 62) The system according to any one of items 51 to 61, wherein the at least one molecule includes a plasmid, DNA, synthetic DNA vector, RNA, nucleic acid-based molecule, antisense oligonucleotide, oligomeric molecule, ribozyme, ribonucleoprotein, CRISPR, recombinant protein, proteolytically targeted chimera, zinc finger nuclease or transcription activator-like effector nuclease, protein and / or material that induces genetic or epigenetic changes in cellular behavior. (Item 63) The cells are located within the retina of the eye, in the system described in any one of items 51 to 62. (Item 64) The system according to item 63, wherein the cells are located within the rod-cone layer of the retina. (Item 65) The system according to item 63, wherein the cells are located within the retinal pigment epithelium of the retina.

[0184] Incorporation by Reference

[0184] All publications, patents, and patent applications mentioned in the present specification are hereby incorporated by reference in the present specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0185]

[0185] In the drawings, which are not necessarily drawn to scale, like numerals may describe similar components in different views. Like numerals with different letter suffixes may represent different instances of similar components. The drawings broadly illustrate, by way of example and not by limitation, the various embodiments discussed in this document. [Figure 1A]

[0186] illustrates an example of a conventional in vitro electroporation system. [Figure 1B]

[0186] illustrates an example of a conventional in vitro electroporation system. [Figure 2]

[0187] illustrates an embodiment of a delivery transition system. [Figure 3A]

[0188] illustrates the direct injection of a plasmid into a target tissue through a needle. [Figure 3B]

[0189] illustrates the delivery of energy from an energy delivery device inserted into the position of the needle of FIG. 3A. [Figure 4A]

[0190] illustrates the direct injection of a plasmid into a target tissue through an energy delivery body. [Figure 4B]

[0191] Figure 4A illustrates the energy delivery from the energy delivery device. [Figure 5]

[0192] The diagram illustrates the locally delivered plasmid while energy is being delivered locally, and, if necessary, while the plasmid is also being delivered locally. [Figure 6]

[0193] The diagram illustrates an energy delivery device comprising a shaft having an energy delivery body near its distal end, wherein the energy delivery body comprises multiple tines. [Figure 7]

[0194] The diagram illustrates an energy delivery device comprising an energy delivery body having a cage shape configured to treat target tissue intraluminally. [Figure 8]

[0195] Another embodiment of an energy delivery device is illustrated, comprising an energy delivery body having a shape configured to treat target tissue intraluminally, wherein the energy delivery body comprises at least two projections, each projection extending radially outward to contact the inner wall of the lumen. [Figure 9]

[0196] Another embodiment of an energy delivery device is illustrated, comprising an energy delivery body having a shape configured to treat target tissue intraluminally, wherein the energy delivery body comprises an expandable member (e.g., an inflatable balloon) on which electrodes are mounted or incorporated. [Figure 10]

[0197] An embodiment of an energy delivery device 102 having a fingertip shape configured such that the delivery body 108 contacts the inner wall of the lumen is shown. [Figure 11A]

[0198] Various stages of an extravasation procedure are illustrated. [Figure 11B]

[0198] Various stages of an embodiment of the extravasation procedure are illustrated. [Figure 11C]

[0198] Various stages of an embodiment of the extravasation procedure are illustrated. [Figure 12A]

[0199] It illustrates an example of a waveform of pulsed electric field (PEF) energy provided by an energy delivery algorithm of a generator used to induce extravasation. [Figure 12B]

[0199] It illustrates an example of a waveform of pulsed electric field (PEF) energy provided by an energy delivery algorithm of a generator used to induce extravasation. [Figure 13]

[0200] It illustrates data generated by various types of delivery of plasmid DNA encoding enhanced green fluorescent protein (EGFP) using PEF energy. [Figure 14]

[0201] It illustrates the data of Figure 13 as average values. [Figure 15A]

[0202] It illustrates an example of a DC current-based waveform provided by an energy delivery algorithm. [Figure 15B]

[0202] It illustrates an example of a DC current-based waveform provided by an energy delivery algorithm. [Figure 15C]

[0202] It illustrates an example of a DC current-based waveform provided by an energy delivery algorithm. [Figure 16]

[0203] It illustrates an embodiment of a special waveform with alternating polarity, wherein the pulse is "sliced", divided or segmented into a series of pulses of the same phase. [Figure 17]

[0204] It illustrates an embodiment of a biphasic waveform, wherein pulses are "sliced", divided or segmented into a series of pulses of the same phase. [Figure 18A]

[0205] It illustrates an embodiment of a waveform having a combination of high short pulses combined with low long pulses. [Figure 18B]

[0205] An embodiment of a waveform having a combination of high and short pulses combined with low and long pulses is shown. [Figure 18C]

[0205] An embodiment of a waveform having a combination of high and short pulses combined with low and long pulses is shown. [Figure 19]

[0206] The diagram illustrates a packet containing a series of pulses grouped together. [Figure 20]

[0207] The diagram illustrates a packet containing a series of biphasic pulses or cycles, each of which has a switch time delay. [Figure 21]

[0208] An embodiment of the waveform is shown, having a series of high-voltage, high-frequency pulses followed by a series of low-voltage, low-frequency pulses, where this combination assists in the transfer of molecules to target cells. [Figure 22]

[0208] An embodiment of the waveform having a series of high-voltage high-frequency pulses followed by a series of low-voltage low-frequency pulses is illustrated, where this combination assists in the transfer of molecules to target cells. [Figure 23]

[0209] Another embodiment of the pulse waveform is illustrated. [Figure 24A]

[0210] An additional embodiment of the waveform provided by algorithm 152 is illustrated, where the pulse includes high-frequency, high-speed oscillation. [Figure 24B]

[0210] An additional embodiment of the waveform provided by algorithm 152 is illustrated, in which the pulse includes high-frequency high-speed oscillation. [Figure 24C]

[0210] An additional embodiment of the waveform provided by algorithm 152 is illustrated, in which the pulse includes high-frequency high-speed oscillation. [Figure 25A]

[0211] The diagram illustrates pressure sensors positioned at various locations along the distal end of an energy delivery device. [Figure 25B]

[0211] The diagram shows pressure sensors positioned at various locations along the distal end of the energy delivery device. [Figure 25C]

[0211] The diagram shows pressure sensors positioned at various locations along the distal end of the energy delivery device. [Figure 26]

[0212] The diagram illustrates embodiments of an energy delivery body and an intraluminal energy delivery device with appropriate operability, which facilitate direct placement and energy delivery to the walls of organs such as the heart. [Figure 27]

[0213] Figure 26 illustrates the position of the coronary arteries relative to the aorta and the position of the energy delivery device. [Figure 28]

[0214] This diagram illustrates a cross-section of the retina near the optic nerve. [Figure 29]

[0215] An embodiment of an energy delivery system configured to deliver energy to the eye area is illustrated. [Figure 30]

[0216] An embodiment of the procedure for forming a subretinal bleb is illustrated. [Figure 31]

[0217] An embodiment of the positioning of an intraocular energy delivery device is illustrated, such that the electrode body is positioned within the subretinal bleb. [Figure 32A]

[0218] This diagram illustrates molecules that have entered the retinal pigment epithelium upon application of a pulsed electric field. [Figure 32B]

[0218] This diagram illustrates molecules that have entered the retinal pigment epithelium when pulsed electric field energy is applied. [Figure 33]

[0219] An embodiment is illustrated in which a molecule is delivered to a subretinal bleb, and an electrode body is positioned within the subretinal bleb for unipolar energy delivery to it. [Figure 34]

[0220] An embodiment is illustrated in which molecules are delivered to a subretinal bleb, and an electrode body is positioned within the vitreous cavity of the eye for unipolar energy delivery to it. [Figure 35]

[0221] An embodiment is illustrated in which molecules are delivered to the vitreous cavity, and an electrode body is positioned in the vitreous cavity for unipolar energy delivery to it. [Figure 36]

[0222] The diagram illustrates molecules and two electrodes positioned within a subretinal bleb, which are positioned in the eye to deliver energy from one to the other in a bipolar manner. [Figure 37]

[0223] The diagram illustrates molecules and two electrodes positioned within the vitreous cavity, with the two electrodes positioned in the eye to deliver energy from one to the other in a bipolar manner. [Figure 38]

[0224] The diagram illustrates molecules and two electrodes positioned in a subretinal bleb, which are positioned in the eye to deliver energy from one to the other in a bipolar manner. [Figure 39]

[0225] The diagram illustrates bipolar energy delivery to the eye, where one electrode is placed in the vitreous cavity, the other electrode is placed in the posterior space of the eyeball, and the molecule is placed in a subretinal bleb. [Figure 40]

[0226] The diagram illustrates bipolar energy delivery to the eye, where one electrode is positioned in a subretinal bleb and the other electrode is positioned in the posterior space of the eye, with the molecule positioned within the subretinal bleb. [Figure 41]

[0227] The diagram illustrates bipolar energy delivery to the eye, where one electrode is positioned in a subretinal bleb, the other electrode is positioned in the posterior space of the eyeball, and the molecule is positioned in the vitreous cavity. [Figure 42]

[0228] The diagram illustrates bipolar energy delivery to the eye, where one electrode is placed in a subretinal bleb and the other electrode is placed in the posterior space of the eye, and molecules are placed within the subretinal bleb and the vitreous cavity. [Figure 43]

[0229] The diagram illustrates an embodiment in which molecules are delivered into the vitreous cavity, a first electrode body is positioned within the vitreous cavity, and a second electrode body is positioned in contact with the cornea. [Figure 44A]

[0230] The illustration shows an embodiment of an energy delivery device configured to deliver a solution, for example, a solution containing molecules, to a target tissue. [Figure 44B]

[0230] An embodiment of an energy delivery device configured to deliver a solution, for example, a solution containing molecules, to a target tissue is shown. [Figure 45]

[0231] An embodiment of an energy delivery device is shown, illustrating a close-up image of its distal end. [Modes for carrying out the invention]

[0186]

[0232] Devices, systems, and methods are provided for delivering molecules, particularly small molecules and / or macromolecules, to cells within the body, especially target cells that directly benefit from the function of those molecules. Such delivery is considered a transfer or biotransfer. Examples of molecules include, to name a few, DNA plasmids, RNA (e.g., messenger RNA (mRNA), small interfering RNA (siRNA), microRNA), oligonucleotides, antisense oligonucleotides (ASOs), proteins and / or materials that induce genetic or epigenetic changes in cellular behavior. In such cases, the molecules are delivered into the target cell using a pulsed electric field (PEF) that delivers the molecules through the cell wall of the target cell so that the desired gene exerts the desired effect within the target cell. Thus, the molecules are delivered to a desired location in the body at a desired time and concentration relative to the delivery of the pulsed electric field for optimal results, as described herein. The molecules can be administered to the body by various methods, such as systemic, localized, and / or direct injection into the region of the target cell. The pulsed electric field energy that delivers molecules into cells is delivered to target cells both in vivo and in situ. Therefore, molecules can be introduced into cells without using viruses or ex vivo methods such as in vitro electroporation.

[0187]

[0233] These devices, systems, and methods are superior to delivery methods such as adeno-associated virus (AAV), a known virus-based gene therapy. AAV has limitations in payload capacity. AAV can only accommodate genes smaller than 4.4 kb, and there is limited space for gene regulatory regions (e.g., promoters). There are more than 300 disease genes that are too large for AAV-based gene therapy. These include genes that cause many relatively common diseases, such as Stargardt disease, Usher 1B and 1D, Leber congenital amaurosis-10 (LCA10), and cystic fibrosis. In contrast, the nonviral delivery methods described herein can accommodate genes larger than 10 kb and have no space limitations. Therefore, many genes that are excluded from use in AAV delivery can be delivered by the devices, systems, and methods described herein. Furthermore, treatment is hindered because many patients have pre-existing anti-AAV antibodies, and immune responses to viral proteins or sequences prevent retreatment. Such immunological considerations do not apply to the nonviral delivery methods described herein, and patients typically exhibit sufficient tolerance to pulsed electric fields. Furthermore, AAV gene therapy can only target tissues and cell types that match existing AAV serotypes. Since pulsed electric field delivery is applicable to all cell types and tissues, such limitations also do not apply to the nonviral delivery methods described herein.

[0188]

[0234] These devices, systems, and methods are superior to delivering pre-transfected cells to the body using in vitro laboratory techniques. As previously mentioned, when transfecting cells in culture for patient treatment, such transfection requires several additional steps. These steps add substantial burden, cost, invasiveness, time, and risk to the delivery of gene therapy.

[0189]

[0235] While the drawbacks of viral and apheresis-based delivery are circumvented by the devices, systems, and methods described herein, direct delivery of molecules to cells within the body using PEF faces several challenges. Such delivery inherently requires the delivery of various devices and components to target locations within the body, which are subject to anatomical and physiological constraints. Devices involved in the delivery of molecules and / or PEF energy for delivery are specifically designed to access target tissues within the body, for example endoscopically, and deliver the desired components to target cells.

[0190]

[0236] Furthermore, the body contains a highly diverse range of target tissues and local environments that are constantly changing in the face of physiological responses, states, and processes. Protocols involved in the delivery of molecules and PEF energy are tailored to maximize transfer in various situations and in the face of changing environments. In some cases, the protocol, and therefore changes in the environment, are induced to benefit the outcome. This is particularly true when delivering and concentrating molecules at desired target locations within the body for transfer to target cells.

[0191]

[0237] Other challenges include delivering PEF energy to target cells in the body in a manner that induces molecular migration while minimizing potential adverse effects on the target cells themselves and / or any surrounding cells. In some cases, PEF energy can be used for tissue ablation rather than molecular migration. Ablation involves cell death, which is substantially the opposite of the desired outcomes of gene therapy and other therapies that rely on cells surviving at least long enough to produce and induce the therapeutic effects of the migrated molecules. In some cases, PEF can kill cells via a series of non-thermal and thermal mechanisms, depending on the waveform and protocol of the PEF used. This is particularly relevant when high voltage is utilized. Therefore, simply increasing the voltage to achieve more migration can be harmful and may lead to unwanted cell death. Thus, numerous variables are carefully optimized and balanced to achieve migration without causing death.

[0192]

[0238] In some cases, device design is optimized to control the unique circumstances of intracellular transfer. When energy is emitted from electrodes on the device (e.g., in a unipolar form using a remote return electrode), cells closest to the electrode may receive high energy levels, which decrease exponentially with distance from the electrode. Thus, depending on the energy band, cells closest to the electrode may die, while distant cells receive the transferred cells, and even further away cells remain unaffected. All of these bands can exist within a few millimeters of the electrode. Increased voltage can exacerbate such cell death, making the transfer of large and / or large total cell numbers difficult. These challenges are specific to intracellular delivery, where laboratory methods using plate electrodes at controlled distances are rarely applicable.

[0193]

[0239] Furthermore, intracellular delivery requires consideration of the systemic environment and potential responses to the delivery procedure. In particular, when using conventional transfection methods designed for in vitro laboratory techniques, unwanted muscle contractions can occur. Such methods require the delivery of long-duration waveforms (e.g., several hundred microseconds to several hundred milliseconds) as part of the transfection procedure. In vivo, this activates motor neurons and skeletal muscles, which can cause patient injury (e.g., falling from a table, movement of the device within the patient's body), pain (e.g., requiring local or general anesthesia), or affect treatment outcomes (e.g., through the transfer of electrodes during the procedure). Neuromuscular palsy agents can help reduce this risk, but this requires general anesthesia and is often insufficient for adequate relaxation of contractions.

[0194]

[0240] The devices, systems, and methods described herein overcome these unique challenges, bringing in vivo PEF transfer to the forefront of clinically valid, translational, and promising therapeutic approaches.

[0195]

[0241] These devices, systems, and methods deliver energy to cells using energy delivery systems. Generally, an energy delivery system comprises a dedicated energy delivery device, a waveform generator, and at least one unique energy delivery algorithm. Additional accessories and devices may be used. For example, in some embodiments, the energy delivery device is delivered through an endoscope, typically an endoscope specific to the anatomical location in which it is used, such as a gastroscopy (upper gastrointestinal endoscopy including the stomach, esophagus, and small intestine (duodenum)), a colonoscope (large intestine), a bronchoscope (lungs), a laryngoscope (larynx), a cystoscope (urinary tract), a duodenoscope (small intestine), a jejunoscopy (digestive system), a ureteroscope (ureters), a hysteroscope (cervix, uterus), etc. In other embodiments, it will be understood that the energy delivery device can be delivered through a catheter, sheath, introducer, needle, or other delivery system.

[0196]

[0242] Intraluminal access allows for the treatment of target tissue from within various lumens in the body. Lumens are the internal spaces of tubular or hollow structures within the body, some examples of which include passages, tubes, conduits, and cavities. Examples of luminal structures include blood vessels, esophagus, stomach, small and large intestines, colon, bladder, urethra, collecting ducts, uterus, vagina, Fallopian duct, ureters, kidneys, renal tubules, spinal canal, spinal cord, and others throughout the body, as well as internal structures of organs such as the lungs, heart, and kidneys, and structures containing those organs. In some embodiments, target tissue is accessed via a nearby luminal structure. In some cases, the energy delivery device advances through various luminal structures or branches of luminal systems to reach the target tissue site. For example, when accessing a target tissue site via a blood vessel, the energy delivery device may be inserted from a distance and advance through various branches of the vascular structure to reach the target site. Similarly, if the luminal structure originates from a natural opening (e.g., nose, mouth, urethra, or rectum), it may enter through that natural opening, and then the energy delivery device may advance through the branching of the luminal system to reach the target tissue location. Alternatively, the luminal structure may be brought near the target tissue via angiotomy or other means. This may be the case when accessing a luminal structure that is not part of a larger system or a luminal structure that is difficult to access by other means.

[0197]

[0243] It will be understood that various anatomical locations can be treated intraluminally using the systems and methods described herein. Examples include the luminal structures themselves, soft tissues of the whole body located near luminal structures, and parenchymal organs accessible from luminal structures (including, but not limited to, the liver, pancreas, gallbladder, kidneys, prostate, ovaries, lymph nodes and lymphatic drainage ducts, the underlying muscular system, bone tissue, brain, eyes, and thyroid gland). It will also be understood that various tissue locations can be accessed percutaneously or by other means.

[0198]

[0244] It is possible to treat target tissue cells at any location throughout the body, some examples of which include cells of the digestive system (e.g., mouth, glands, esophagus, stomach, duodenum, jejunum, ileum, intestine, colon, rectum, liver, gallbladder, pancreas, anal canal, etc.), cells of the respiratory system (e.g., nasal cavity, pharynx, larynx, trachea, bronchi, lungs, etc.), cells of the urinary system (e.g., kidneys, ureters, bladder, urethra, etc.), and cells of the reproductive system (e.g., genitals, ovaries, fallopian tubes, uterus, cervix, vagina, testes, epididymis, vas deferens, seminal vesicles, prostate). Examples include cells of the glands, penis, scrotum, etc., cells of the endocrine system (e.g., pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland), cells of the circulatory system (e.g., heart, arteries, veins, etc.), cells of the lymphatic system (e.g., lymph nodes, bone marrow, thymus, spleen, etc.), cells of the nervous system (e.g., brain, spinal cord, nerves, ganglia, etc.), cells of the eye (e.g., retina, macula, cone layers, retinal pigment epithelium, optic nerve, choroid, sclera, etc.), cells of the muscle system (e.g., muscle cells, etc.), and cells of the skin (e.g., epidermis, dermis, subcutaneous tissue, etc.).

[0199]

[0245] The energy delivery device delivers energy provided by the waveform generator according to at least one unique energy delivery algorithm. In some embodiments, it will be understood that the energy delivery device also delivers molecules. However, in other embodiments, molecules are delivered by a separate device such as an IV, catheter, or needle injection. If necessary, molecules may be delivered by both the energy delivery device and the separate device. Examples of embodiments of dedicated energy delivery devices provided herein mainly focus on unipolar energy delivery, but it will be understood that bipolar or multipolar mechanisms may also be used.

[0200]

[0246] Figure 2 illustrates an embodiment of an energy delivery system 100 comprising a dedicated energy delivery device 102, a return electrode 115, and a waveform generator 104. In this embodiment, the target tissue is located within the liver L of patient P. In this embodiment, the energy delivery device 102 comprises a flexible, elongated shaft having a distal end that can be advanced luminally to the target tissue within the liver L. As shown, the distal end of the delivery device 102 passes through the mouth M, down the esophagus E, advances into the stomach S, passes through the stomach wall, and enters the liver L. In some embodiments, the distal end has a distal tip 103 configured to penetrate the stomach wall and / or the liver L. In other embodiments, a passage through the stomach wall is formed using a separate instrument which is subsequently removed, and the energy delivery device 102 having a non-invasive tip can pass through that passage.

[0201]

[0247] In this embodiment, molecule 110 is delivered systemically intravenously using an IV bag 112. This distributes molecule 110 throughout the patient P's body, typically including target tissue within the liver L. In other embodiments, it will be understood that molecule 110 is delivered locally. In such embodiments, molecule 110 may be delivered to upstream vascular structures in the arterial system leading to the target organ or target tissue region. The molecule 110 then travels to the target region through the downstream arterial circulation. If a bolus injection of molecule 110 is provided, a rapid increase in molecule 110 will enter the target tissue. However, if molecule 110 is delivered over time, such as by the use of an infusion pump, a stable and sustained level of molecule 110 can be achieved in the target tissue. In other embodiments, it will be understood that molecule 110 is delivered by direct injection into the target tissue. In such embodiments, an injection device is inserted into or near the target tissue, such as within the parenchymal tissue of the target organ region, and a solution containing affector genetic material is injected. A solution of this molecule can reach the target region or volume of delivery by allowing distribution through the parenchyma and interstitial space for a certain period of time. It will be understood that any combination of systemic delivery, regional delivery, and local delivery may be used as alternatives.

[0202]

[0248] The pulsed electric field energy is delivered to the target tissue through the distal end of the delivery device 102. The proximal end of the delivery device 102 is electrically connected to the waveform generator 104. In some embodiments, the generator 104 is also connected to an external cardiac monitor (not shown) to enable energy delivery in coordination with cardiac signals sensed from the patient P.

[0203]

[0249] In this embodiment, the energy delivery device 102 is designed to be unipolar, with the distal end of the delivery device 102 having a delivery electrode, and the return electrode 115 is positioned on the skin outside the body, typically on the thigh (as shown), waist, or back.

[0204]

[0250] A pulsed electric field (PEF) is provided by a generator 104 and delivered to the tissue through an energy delivery body 108 positioned on, within, or near the targeted tissue region. In some embodiments, it will be understood that the energy delivery body 108 is positioned in contact with a conductive material that is also in contact with the targeted tissue. Such a solution may include an isotonic or hypertonic solution. Electrical pulses are then delivered through the energy delivery body 108 near the target tissue. These electrical pulses are provided by at least one energy delivery algorithm 152. The algorithm 152 specifies various parameters of the signal that contribute to the overall shape of the waveform, for example, energy amplitude (e.g., voltage), as well as the duration of the applied energy, which consists of the number of pulses, pulse width, and delay between pulses. In some embodiments, one or more energy delivery bodies are small and tend to dissipate a large amount of energy around the electrodes. Therefore, optimal energy delivery is desired. In some embodiments, a large DC link capacitance by a transistor half-bridge is utilized to provide efficient delivery pulses in such cases. In some cases, this is preferable with respect to pulse voltages delivered by a power amplifier (with limited bandwidth) or an exponential attenuation generator. In some embodiments, this may include a feedback loop based on sensor information and an auto-shutoff specification.

[0205]

[0251] As will be described in later sections, biphasic pulses may be used in some embodiments. In such embodiments, additional parameters may include the switching time between polarities in the biphasic pulse and the dead time between biphasic cycles. Biphasic waveforms may be used to reduce muscle stimulation in a patient. This is particularly important in applications where even slight movement of the energy delivery body may negate the therapeutic effect or lead to adverse consequences. Biphasic waveforms require rapid changes in the phase / polarity of the signal to minimize nerve activation during polarity changes. Multiple high-speed switching elements (e.g., MOSFETs, IGBT transistors) are desirable and may be employed, for example, in an H-bridge or full-bridge configuration.

[0206]

[0252] Referring back to Figure 1, in this embodiment, the generator 104 includes a user interface 150, one or more energy delivery algorithms 152, a processor 154, a data storage / retrieval unit 156 (e.g., memory and / or database), and an energy storage subsystem 158 that generates and stores the energy to be delivered. In some embodiments, one or more capacitors are used for energy storage / delivery, but any other suitable energy storage element may be used. Furthermore, one or more communication ports may be included.

[0207]

[0253] In some embodiments, the generator 104 comprises three subsystems: 1) a high-energy storage system, 2) a high-voltage medium-frequency switching amplifier, and 3) a system controller, firmware, and user interface. The generator takes in an alternating current (AC) power supply and powers multiple direct current (DC) power supplies. The generator's controller can cause the DC power supplies to charge the high-energy capacitor storage bank before initiating energy delivery. In some embodiments, at the start of energy delivery, the generator's controller, high-energy storage bank, and biphase pulse amplifier can operate simultaneously to generate a high-voltage medium-frequency output.

[0208]

[0254] It will be recognized that numerous generator electrical architectures can be employed to implement energy delivery algorithms. In particular, in some embodiments, advanced switching systems are used that allow pulsed electric field circuits to be directed separately from the same energy storage and high-voltage delivery system to the energy delivery electrodes. Furthermore, generators employed in advanced energy delivery algorithms using rapidly changing pulse parameters (e.g., voltage, frequency, etc.) or multiple energy delivery electrodes can utilize modular energy storage and / or high-voltage systems, promoting a highly customizable waveform and geographic pulse delivery paradigm. It should be further recognized that the electrical architectures described herein are merely examples, and the system for delivering pulsed electric fields may or may not include additional components such as switching amplifiers.

[0209]

[0255] The user interface 150 may include a touchscreen and / or more conventional buttons that allow the operator to input patient data, select a treatment algorithm (e.g., an energy delivery algorithm 152), initiate energy delivery, view records stored in the storage / retrieval unit 156, and / or communicate with the generator 104 in other ways.

[0210]

[0256] In some embodiments, the user interface 150 is configured to receive operator-defined inputs. Operator-defined inputs may include the duration of energy delivery, the pulses of energy delivery, power, and / or one or more other timing aspects of the operating mode, or a combination thereof. Examples of operating modes include (but are not limited to) system startup and self-diagnosis, operator input, algorithm selection, pre-processing system status and feedback, energy delivery, display or feedback after energy delivery, review and / or download of processing data, software updates, or any combination or partial combination thereof.

[0211]

[0257] In some embodiments, the processor 154 modifies and / or switches the energy delivery algorithm, monitors energy delivery and any sensor data, and responds to the monitored data via a feedback loop, among other tasks. In some embodiments, the processor 154 is configured to execute one or more algorithms for running a feedback control loop based on one or more measured system parameters (e.g., current), one or more measured tissue parameters (e.g., impedance), and / or a combination thereof.

[0212]

[0258] The data storage / retrieval unit 156 stores data (e.g., data related to the procedures to be delivered) and, if necessary, can be downloaded by connecting a device (e.g., a laptop or thumb drive) to the communication port. In some embodiments, the device has local software used to instruct the download of information (e.g., instructions stored in the data storage / retrieval unit 156 and executable by the processor 154). In some embodiments, the user interface 150 allows the operator to select data and download it to a device and / or system (e.g., a computer device, tablet, mobile device, server, workstation, cloud computing device / system, etc., but not limited to these). The communication port, which can enable wired and / or wireless connections, can also enable data uploads (e.g., uploading custom algorithms or providing software updates) in addition to the data downloads described above.

[0213]

[0259] As described herein, various energy delivery algorithms 152 (for example, those stored in memory or the data storage / retrieval unit 156) can be programmed into or pre-programmed for the generator 104. Alternatively, energy delivery algorithms can be added to the data storage / retrieval unit to be executed by the processor 154. Each of these algorithms 152 may be executed by the processor 154.

[0214]

[0260] In some embodiments, the energy delivery device 102 includes one or more sensors that can be used to measure, to name a few, temperature, impedance, resistance, capacitance, conductivity, pH, optical properties (coherence, echo brightness, fluorescence), dielectric constant or photodielectric constant, and / or conductance. In some embodiments, one or more electrodes act as one or more sensors. In other embodiments, one or more sensors are separate from the electrodes. Sensor data can be used to provide direct feedback that can be used to plan treatment, monitor treatment, and / or modify the energy delivery algorithm 152 via the processor 154. For example, impedance measurements can be used not only to determine the initial amount to be applied, but also to determine whether further energy delivery is necessary.

[0215]

[0261] In some embodiments, it will be understood that the system 100 includes an automated treatment delivery algorithm that dynamically responds to inputs such as temperature, impedance at various voltages or AC frequencies, duration or other timing aspects of energy delivery pulses, treatment power and / or system status, and adjusts and / or terminates delivery in accordance with those inputs.

[0216] Device Embodiment

[0262] Energy can be delivered by various energy delivery devices 102. Typically, the energy delivery device 102 comprises a flexible, elongated shaft having a distal end that can be advanced with the body to target tissue, and at least one energy delivery body 108 positioned near the distal end. The energy delivery body 108 comprises one or more electrodes that deliver PEF energy to the target tissue.

[0217]

[0263] As previously mentioned, in some embodiments, the energy delivery device 102 delivers PEF energy, and the molecule 110 is delivered by a separate device such as an IV, catheter, or needle injection. Figure 3A illustrates the direct injection of molecule 110 into target tissue through a needle 500. The target tissue is shown as cell C (not to exact scale). The needle 500 is inserted into or near the target tissue so that the target tissue can be immersed in the injected molecule 110. In this embodiment, the needle 500 is then withdrawn, and the molecule 110 remains for in vivo distribution. Referring to Figure 3B, the distal end of the delivery device 102 is then inserted into the target tissue so that the energy delivery body 108 is preferably positioned in or near the target tissue. In this embodiment, the energy delivery body 108 consists of a single electrode. PEF energy is then delivered from the energy delivery body 108 to the target tissue, as indicated by the dashed line 502. This PEF energy causes molecule 110 to migrate to cell C.

[0218]

[0264] In some embodiments, the molecule 110 and energy are delivered by an energy delivery device 102. Figures 4A to 4B illustrate the energy delivery device 102 having a needle-shaped energy delivery body 108. The needle-shaped tip can penetrate like a needle and deliver the molecule 110 from its lumen. Furthermore, the energy delivery body 108 is electrically insulated by an insulating layer 504, except for the needle-shaped tip that acts as an electrode. Figure 4A illustrates the direct injection of molecule 110 into target tissue via the energy delivery body 108. Here again, the target tissue is illustrated as cell C (not to exact scale). The tip is inserted into or near the target tissue so that the target tissue can be immersed in the injected molecule 110 and, if necessary, so that the molecule remains for in vivo distribution. Referring to Figure 4B, PEF energy is then delivered from the energy delivery body 108 to the target tissue, as indicated by the dashed line 502. This PEF energy causes the molecule 110 to migrate into cell C.

[0219]

[0265] Figure 5 illustrates how energy is delivered locally while molecules 110 are delivered locally (molecules 110 are additionally delivered locally as needed). Here, molecules 110 are delivered by a separate device, such as a catheter 501, positioned within a vascular structure V nourishing the target tissue region. Thus, molecules 110 are delivered locally to the target tissue region. The energy delivery device 102 is inserted into the target tissue region by a different approach. Here, the energy delivery device 102 comprises an energy delivery body 108 having a needle shape. The needle-shaped tip can penetrate like a needle. In some embodiments, molecules 110 can be delivered from its lumen. In this embodiment, the energy delivery body 108 is electrically insulated by an insulating layer 504, except for the needle-shaped tip that acts as an electrode. The tip is inserted into or near the target tissue, and then PEF energy is delivered from the energy delivery body 108 to the target tissue as indicated by the dashed line 502. This PEF energy allows molecule 110 to translocate into the target cell.

[0220]

[0266] Figure 6 illustrates an energy delivery device 102 comprising a shaft 106 having an energy delivery body 108 near its distal end, wherein the energy delivery body 108 comprises a plurality of tines 600. Typically, the tines 600 have a pointed shape to penetrate tissue. Similarly, the tines 600 typically extend laterally outward from the shaft 106, and in some embodiments, the tines 600 unfold circumferentially around the shaft 106. In some embodiments, it will be understood that the tines 600 unfold from the sides of the shaft 106, such as being aligned in a row. In some embodiments, the tines 600 extend by the same distance from the shaft 106, and in other embodiments, the tines 600 extend by varying distances. In some embodiments, it will be understood that the extension of at least a portion of the tines 600 from the shaft 106 is adjustable.

[0221]

[0267] Typically, each tine 600 delivers molecules 110 and / or energy from it. In some embodiments, the molecules 110 are delivered from the tip 601 of the tine 600, and in other embodiments, the molecules 110 are delivered from a delivery port 602 along the tine 600. In some embodiments, the tines 600 are energizable collectively (e.g., to act as a single electrode), or at least some of the tines 600 are energizable individually (e.g., to act as a bipolar pair, or to act as a selectable single electrode, including acting in a group). In some embodiments, one or more tines 600 deliver different energies (e.g., energies generated from different energy delivery algorithms 152) and / or different types of molecules 110.

[0222]

[0268] In this embodiment, the shaft 106 has three sections: a first section 106a, a second section 106b, and a third section 106c. As illustrated in Figure 6, the first section 106a is distal to the second section 106b, and the second section 106b is distal to the third section 106c. Each section 106a, 106b, and 106c may or may not be insulated to create various different electrode combinations. This may allow for various shapes of electric fields and / or direct the electric field in a desired direction. In some embodiments, it will also be understood that at least a portion of at least one tine 600 is insulated to direct the energy emitted therefrom. Overall, the tines 600 often allow a single-location energy delivery device 102 to deliver molecules 110 and / or energy to a larger volume of target tissue than a device 102 having an energy delivery device 108 with a single needle.

[0223]

[0269] In some embodiments, the first section 106a acts as an energy delivery body 108, and one or more tines 600 also act as energy delivery bodies 108. Each of the different energy delivery bodies 108 may deliver the same type of energy or different types of energy, and similarly, the energy delivery bodies 108 may act in groups. In some embodiments, the tines 600 extend beyond the first section 106a. In some embodiments, the tines 600 extend the same distance from the shaft 106 (relative to the first section 106a), and in other embodiments, the tines 600 extend different distances (relative to the first section 106a). In some embodiments, the first section 106a acts as an energy delivery body 108, and one or more tines 600 act as conduits for the delivery of molecules 110.

[0224]

[0270] Figure 7 illustrates an energy delivery device 102 comprising an energy delivery body 108 having a cage shape configured to treat target tissue intraluminally. Here, the target tissue includes cells C located near the wall of a body lumen W, particularly the wall that circumferentially surrounds the body lumen at least partially. In this embodiment, the energy delivery body 108 consists of a plurality of wires or ribbons 120 that form a helical cage acting as an electrode. In some embodiments, the energy delivery body 108 is self-expanding and delivered to the targeting region in a folded configuration. This folded configuration can be achieved, for example, by covering the energy delivery body 108 with a sheath. Self-expansion of the energy delivery body 108 is enabled by contracting the sheath or advancing the energy delivery body 108 from the sheath. In other embodiments, the energy delivery device 102 comprises a handle having an energy delivery body operating knob, where the movement of the knob causes expansion or contraction / folding of the cage electrode. The cage electrode is expandable within a body lumen or passage (naturally occurring or created in the body) so as to contact at least a portion of the wall W of the body lumen. The molecule 110 is delivered from the energy delivery device 102, such as through the distal end port 510 and / or various side ports 512 along the shaft 106 of the device 102, such as within the cage electrode, as illustrated in Figure 7. The target tissue can be immersed in the molecule 110, and if necessary, the molecule 110 can remain for in vivo distribution. PEF energy is then delivered to the target tissue from the energy delivery body 108, as indicated by the dashed line 502. This PEF energy causes the molecule 110 to migrate to cells C.

[0225]

[0271] Figure 8 illustrates another embodiment of the energy delivery device 102, comprising an energy delivery body 108 having a shape configured to treat target tissue intraluminally. In this embodiment, the energy delivery body 108 comprises at least two projections 514, each projection extending radially outward to contact the inner wall W of the lumen. A single projection may be present, but it will be understood that typically two projections exert substantially opposing forces on the lumen wall. In the embodiment of Figure 8, three projections 514 are present. In some embodiments, each projection 514 is formed of a wire or ribbon, which acts as an electrode and is bent or arched radially outward from the longitudinal axis or shaft 106 of the delivery device 102. In this embodiment, the projections 514 act collectively as a single electrode. However, in other embodiments, one or more projections 514 are independently energizable to act as multiple electrodes (e.g., as one or more bipolar pairs). The projection 514 may be made of various suitable materials (e.g., stainless steel, spring steel, or other alloys) to act as an electrode, and may be, for example, a round wire or ribbon. In some embodiments, a portion of the projection 514 is insulated with an insulating segment such as a polymer (e.g., PET, polyether block amide, polyimide). For example, in some embodiments, at least a portion of the proximal and distal ends of the energy deliverer 108 is insulated to direct the energy laterally toward the wall W.

[0226]

[0272] In some embodiments, the energy delivery body 108 in Figure 8 is self-expanding and delivered to the targeting region in a folded configuration. The projections arch outward during expansion within the body lumen or passage (naturally occurring or created in the body) so as to contact at least a portion of the wall W of the body lumen. The molecule 110 is delivered from the energy delivery device 102, such as via the port 516 in the energy delivery body 108 as illustrated in Figure 8. The target tissue can be immersed in the molecule 110, and if necessary, the molecule 110 can remain for in vivo distribution. PEF energy is then delivered from the energy delivery body 108 to the target tissue as indicated by the dashed line 502. This PEF energy causes the molecule 110 to migrate to the cell C.

[0227]

[0273] Figure 9 illustrates another embodiment of the energy delivery device 102, comprising an energy delivery body 108 having a shape configured to treat target tissue intraluminally. In this embodiment, the energy delivery body 108 comprises an expandable member 518 (e.g., an inflatable balloon) on which electrodes 520 are mounted or incorporated. The energy delivery body 108 is delivered to the target area in a folded configuration. In this embodiment, the electrodes 520 have a pad shape with a relatively large surface area and a thin cross-section. Its pad shape provides a larger surface area than other shapes, such as a wire shape. Each electrode 520 is connected to a conduction wire 522 that electrically connects the electrode 520 to the generator. In this embodiment, three electrodes 520 are visible, but it will be understood that additional electrodes may be present around the expandable member 518. Any number of electrodes 520 may be present, and it will be understood that they act as a single electrode or act independently or in combination. The arrangement of electrodes 520 and / or selective energization of electrodes 520 can direct energy to a specific target location. In some embodiments, electrodes 520 consist of flexible circuit pads or other materials attached to or formed on the expandable member 518. In some embodiments, electrodes 520 are distributed radially around the outer circumference of the expandable member 518 and / or longitudinally along the length of the expandable member 518. Such designs can facilitate improved deployment and retraction quality and easier user operation and fit of the introducer to the lumen.

[0228]

[0274] When expanding the expandable member, one or more electrodes 520 are positioned to contact at least a portion of the lumen wall W. The molecule 110 is delivered from the energy delivery device 102 via the distal end port 510, as illustrated in Figure 9. The target tissue can be immersed in the molecule 110, and if necessary, the molecule 110 can remain for in vivo distribution. PEF energy is then delivered to the target tissue from the energy delivery body 108, as indicated by the dashed line 502. This PEF energy causes the molecule 110 to migrate to cells C.

[0229]

[0275] Figure 10 illustrates another embodiment of the energy delivery device 102. Here, the energy delivery body 108 has a fingertip shape configured to contact the inner wall W of a lumen. In this embodiment, the energy delivery device 102 has an elongated shaft 106 and a fingertip electrode 530 positioned at its distal end. The fingertip electrode 530 can be positioned in contact with a portion of the lumen wall W near the target tissue cell C. The molecule 110 can be delivered by any preferred method, for example, systemically, locally, or locally, for example, by injection through a separate device or the energy delivery device 102. Figure 10 illustrates the delivery of molecule 110 via the fingertip electrode 530. When the fingertip electrode 530 is energized, PEF energy is directed towards the cell C, as indicated by the dashed line 502. This PEF energy causes molecule 110 to transfer to the cell C.

[0230]

[0276] In some embodiments, it will be understood that PEF energy is delivered to a conductive fluid (e.g., blood, saline solution, etc.) in contact with the target tissue. Therefore, the energy can pass through that conductive fluid to the target tissue for transfer. In other embodiments, the delivery of energy to the conductive fluid facilitates the transfer of genetic material from the fluid itself to cells, such as the transfer of genetic material to leukocytes in the blood.

[0231] molecule

[0277] As previously stated, the devices, systems, and methods are provided for delivering molecules 110, particularly small molecules and / or macromolecules, to cells in the body (e.g., target cells that directly receive therapeutic benefits from the function of those molecules). Such therapeutic benefits may be in the treatment of various disorders.

[0232]

[0278] In some embodiments, the disorder includes coagulation disorders such as hemophilia (e.g., hemophilia A or hemophilia B), von Willebrand disease, factor XI deficiency, fibrinogen disorder, or vitamin K deficiency. The coagulation disorder may be characterized by mutations in genes encoding fibrinogen, prothrombin, factor V, factor VII, factor VIII, factor X, factor XI, factor XIII, or enzymes involved in their post-translational modification, or enzymes involved in vitamin K metabolism. In some embodiments, the coagulation disorder is characterized by mutations in FGA, FGB, FGG, F2, F5, F7, F10, F11, F13A, F13B, LMAN1, MCFD2, GGCX, or VKORC1.

[0233]

[0279] In some embodiments, disorders include neurological disorders, such as neurodegenerative diseases. In some embodiments, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, or multiple sclerosis. In some embodiments, neurodegenerative diseases include autoimmune diseases of the central nervous system (CNS), such as multiple sclerosis, encephalomyelitis, paraneoplastic syndromes, autoimmune inner ear diseases, or oculoclonus myoclonus syndrome. Neurological disorders may include cerebral infarction, spinal cord injury, central nervous system disorders, neuropsychiatric disorders, or channel diseases (e.g., epilepsy or migraine). Neurological disorders may include anxiety disorders, mood disorders, childhood disorders, cognitive impairments, schizophrenia, substance-related disorders, or eating disorders. In some embodiments, neurological disorders are symptoms of cerebral infarction, stroke, traumatic brain injury, or spinal cord injury.

[0234]

[0280] In some embodiments, the disorders include lysosomal storage disorders, such as Tay-Sachs disease, Gaucher disease, Fabry disease, Pompe disease, Niemann-Pick disease, or mucopolysaccharidosis (MPS).

[0235]

[0281] In some embodiments, the disorder includes cardiovascular disorders, such as degenerative heart disease, coronary artery disease, ischemia, angina pectoris, acute coronary syndrome, peripheral vascular disease, peripheral artery disease, cerebrovascular disease, or atherosclerosis. The cardiovascular disorder may be a degenerative heart disease selected from the group consisting of ischemic cardiomyopathy, conduction disorders, and congenital defects.

[0236]

[0282] In some embodiments, the disorder includes immune disorders, such as autoimmune disorders. Autoimmune disorders may include type 1 diabetes, multiple sclerosis, rheumatoid arthritis, lupus, encephalomyelitis, paraneoplastic syndromes, autoimmune inner ear diseases, or ocular clonus myoclonus syndrome, autoimmune hepatitis, uveitis, autoimmune retinopathy, neuromyelitis optica, psoriatic arthritis, psoriasis, myasthenia gravis, chronic Lyme disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, peripheral neuropathy, fibromyalgia, Hashimoto's thyroiditis, ulcerative colitis, or Kawasaki disease.

[0237]

[0283] In some embodiments, the disorder includes liver diseases, such as hepatitis, Alagille syndrome, biliary atresia, liver cancer, cirrhosis, cystic disease, Calori syndrome, congenital liver fibrosis, fatty liver, galactosemia, primary sclerosing cholangitis, hypertyrosinemia, glycogen storage disease, Wilson's disease, or endocrine deficiency. Liver diseases may include liver cancers, such as hepatocellular hyperplasia, hepatocellular adenoma, focal nodular hyperplasia, or hepatocellular carcinoma.

[0238]

[0284] In some embodiments, the impairment includes cancers, such as hematological cancers (e.g., acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic myeloid leukemia, Hodgkin's disease, multiple myeloma and non-Hodgkin lymphoma) or solid tissue cancers (e.g., liver cancer, kidney cancer, breast cancer, gastric cancer, esophageal cancer, stomach cancer, intestinal cancer, colorectal cancer, bladder cancer, head and neck cancer, skin cancer or brain cancer).

[0239]

[0285] In some embodiments, the disorder includes recessive inheritance disorders. In some embodiments, the disorder is a Mendelian inheritance disorder.

[0240]

[0286] In some embodiments, the disorder includes ocular disorders such as retinal dystrophy (e.g., Mendelian retinal dystrophy). Retinal dystrophy may include Leber congenital amaurosis (LCA), Stargardt disease, pseudoxanthoma elasticum, rod-cone dystrophy, exudative vitreoretinopathy, Joubert syndrome, CSNB-1C, age-related macular degeneration, retinitis pigmentosa, Stickler syndrome, microcephaly and chorioretinopathy, retinitis pigmentosa, CSNB2, Usher syndrome, or Wagner syndrome.

[0241]

[0287] In some embodiments, the molecules 110 delivered by the devices, systems, and methods described herein include synthetic DNA vectors, e.g., those described in the application WO2019178500, publication number March 15, 2019, entitled "Synthetic DNA Vectors and Methods of Use" (which is incorporated herein by reference in its entirety for all purposes). Such synthetic DNA vectors include non-viral DNA vectors, e.g., vectors that induce long-term transduction into quiescent cells (e.g., post-mitotic cells) in a manner similar to AAV vectors. In some embodiments, such non-viral DNA vectors are developed in vitro (e.g., cell-free) systems that synthetically generate circular AAV-like DNA vectors (e.g., DNA vectors containing terminal repeat sequences such as DD elements) by isothermal rolling circle amplification and ligation-mediated circulation (e.g., as opposed to bacterial expression and site-directed recombination). Such developments enable scalability and improved manufacturing efficiency in the production of circular AAV-like DNA vectors. Furthermore, the vectors produced by these methods are designed to overcome many of the problems associated with plasmid DNA vectors, e.g., Lu et al., Mol.Ther. 2017, 25(5):1187-98 (which is incorporated herein by reference in its entirety). For example, transcriptional silencing can be reduced or eliminated by eliminating or reducing the presence of CpG islands and / or bacterial plasmid DNA sequences (e.g., RNAPII arrest sites), thereby improving the persistence of heterologous genes. In addition, the risk of stimulating the host immune system is reduced by eliminating the presence of immunogenic components (e.g., bacterial endotoxins, DNA, RNA, or bacterial signatures such as CpG motifs). Such advantages are particularly beneficial in the treatment of certain disorders, such as retinal dystrophy (e.g., Mendelian retinal dystrophy).

[0242]

[0288] Therefore, such vectors include synthetic DNA vectors that (i) substantially lack bacterial plasmid DNA sequences (e.g., RNAPII arrest sites, replication origins, and / or resistance genes) and other bacterial signatures (e.g., immunogenic CpG motifs), and / or (ii) can be synthesized and amplified entirely in vitro (e.g., do not require replication in bacteria, and do not require bacterial replication origins and bacterial resistance genes). In some embodiments, these vectors include a double D (DD) element characteristic of AAV vectors. This makes it possible to transduce a DNA vector containing heterologous genes that behave like AAV viral DNA (e.g., have low transcriptional silencing and high persistence) into target cells without requiring the virus itself.

[0243]

[0289] In some embodiments, molecule 110 includes nucleic acid-based molecules, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), oligonucleotides, antisense oligonucleotides (ASOs), microRNA (miRNA), decoy DNA, ribozymes, morpholino, and plasmids. RNA interference using small inhibitory RNA (siRNA) allows for downregulation of mRNA levels by cellular nucleases activated upon detection of sequence homology between the siRNA and the respective mRNA molecule. Therefore, in some embodiments, siRNA is used to silence genes involved in the pathogenicity of various diseases associated with a known genetic background. In some embodiments, molecule 110 includes patisirane, an siRNA-based drug approved by the FDA for the treatment of polyneuropathy in individuals with hereditary transthyretin-mediated amyloidosis. For siRNA to function, it must be present in the target cell of interest. This means that the siRNA must be transported to the tissue in the body where the target cell resides, and then it must cross the cell membrane. These requirements are generally referred to as the "delivery" of siRNA to the desired location. Since siRNA is a negatively charged molecule and does not naturally pass through the outer membrane of cells, conventional delivery methods have proven difficult. The devices, systems, and methods described herein overcome these delivery difficulties and deliver siRNA to target cells.

[0244]

[0290] In some embodiments, molecule 110 contains microRNA (miRNA). miRNA is a class of small non-coding RNAs, approximately 22 nt in length, that are involved in regulating gene expression at the post-transcriptional level by degrading their target mRNA and / or inhibiting its translation.

[0245]

[0291] In some embodiments, molecule 110 contains an antisense oligonucleotide (ASO). The ASO is a synthetic DNA oligomer that hybridizes to a target RNA in a sequence-specific manner. In some embodiments, the ASO is delivered to inhibit gene expression, to modulate the splicing of precursor messenger RNA, or to inactivate microRNA. Chemically modified nucleotides, such as phosphorothioates, 2'-O-methyl RNA, or roq nucleic acids, may be used to stabilize the ASO against nuclease degradation, as they confer nuclease resistance. In some embodiments, the ASO is delivered with optimized delivery, specificity, affinity, and enhanced nuclease resistance, and with low toxicity.

[0246]

[0292] Examples of ASOs include (1) homivirsen for the treatment of CMV retinitis in AIDS patients, (2) mipomersen for the treatment of familial hypercholesterolemia, (3) defibrotide for the treatment of hepatic veno-occlusive disease, (4) eteplirsen for the treatment of Duchenne muscular dystrophy, (5) pegaptanib for the treatment of neovascular age-related macular degeneration, and (6) nusinersen for the management of spinal muscular atrophy.

[0247]

[0293] In some embodiments, molecule 110 contains oligomeric molecules such as phosphorodiamidate morpholino oligomers (PMOs) (also known as morpholino), which are a type of oligomeric molecule used to modify gene expression and knock down gene function. Morphorinos are typically 25 nucleotides long and bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base pairing. Morphorino oligos specifically bind to their selected DNA or RNA target sites, blocking access to those sites by cellular components. This property can be used to block translation, block splicing, block microRNAs (miRNAs) or their targets, and block ribozyme activity. Their molecular structure consists of DNA bases attached to a methylenemorpholine ring backbone linked via phosphorodiamidate groups. The uncharged backbone of morpholino oligos is not recognized by enzymes and is therefore completely stable against nucleases. In some embodiments, eteplirsen, a morpholino-based drug that can be used to treat several mutations causing Duchenne muscular dystrophy (DMD), is delivered. In other embodiments, golodirsen, a morpholino-based drug, is delivered for the treatment of DMD.

[0248]

[0294] In some embodiments, molecule 110 includes ribozymes (ribonucleic acid enzymes), which are naturally occurring RNA molecules that catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. In some embodiments, molecule 110 includes synthetic ribozymes, such as those designed to inhibit protein production through the specific cleavage of disease-causing mRNA. Another application of ribozyme therapy is the inhibition of RNA-based viruses, such as HIV, hepatitis C virus, SARS coronavirus (SARS-CoV), adenovirus, and influenza A and B viruses.

[0249]

[0295] In some embodiments, molecule 110 contains ribonucleoprotein (RNP). RNP is a complex formed between RNA and an RNA-binding protein. For example, by combining purified Cas9 protein with guide RNA, an RNP complex can be formed that is delivered to cells for rapid and highly efficient genome editing. Because RNP remains in cells for a short time and is used in minimal doses, it is less toxic and causes less off-target editing compared to other methods. Furthermore, since the RNP complex does not contain DNA, there is no risk of insertional mutagenesis.

[0250]

[0296] In some embodiments, the molecule 110 delivered by the devices, systems, and methods described herein contains clustered, regularly arranged, short repeating palindromic sequence repeat (CRISPR) DNA sequences called CRISPR. These DNA sequences were originally observed in bacteria, with "spacer" DNA sequences between the repeats that perfectly match the viral sequence. Subsequently, it was discovered that upon viral infection, bacteria transcribe these DNA elements into RNA. This RNA protects against the virus by guiding a nuclease (a protein that cuts DNA) to the viral DNA and cleaving it. This nuclease is named "Cas," meaning "CRISPR-associated."

[0251]

[0297] In 2012, researchers demonstrated that it was possible to construct RNA that guides Cas nucleases (Cas9 was the first to be used) to any DNA sequence. This so-called guide RNA can also be crafted to be specific to only that single sequence, thus improving the likelihood that the DNA will be cleaved at that site and not anywhere else in the genome. Further testing revealed that this system works quite well in all types of cells, including human cells.

[0252]

[0298] Using CRISPR / Cas, targeted genes can be disrupted, and by adding a DNA template to the mixture, new sequences can be inserted at the desired precise point. This method has been used to develop animal models with specific genomic mutations. Furthermore, in the case of human diseases with known mutations, such as cystic fibrosis, it is theoretically possible to insert DNA that corrects these mutations. However, delivering large quantities of CRISPR / Cas material to mature cells using conventional methods such as viral vectors has been difficult. However, the devices, systems, and methods described herein overcome these difficulties by enabling the delivery of molecules 110 containing CRISPR / Cas material to cells.

[0253]

[0299] In some embodiments, molecule 110 contains recombinant proteins. Such therapeutic proteins have been developed using recombinant DNA technology to treat a wide variety of diseases, including cancer, autoimmune / inflammatory diseases, infections with infectious pathogens, and genetic disorders.

[0254]

[0300] In some embodiments, molecule 110 contains a proteolytic targeting chimera (PROTAC). PROTAC is a small molecule capable of removing specific unwanted proteins. PROTAC consists of two covalently linked protein-binding molecules: one that can engage with an E3 ubiquitin ligase, and the other that binds to the target protein for degradation. Once the E3 ligase is recruited to the target protein, ubiquitination occurs, followed by degradation of the target protein by the proteasome. Currently, PROTAC is successfully used to degrade various types of target proteins associated with a wide range of diseases, including cancer, viral infections, immunodeficiencies, and neurodegenerative diseases. PROTAC offers various advantages in cancer treatment, including overcoming drug resistance and degrading protein targets that were previously considered "undrug-resistant." Currently, only 20-25% of known protein targets can be targeted using conventional drug discovery techniques. Proteins lacking catalytic activity and / or proteins with catalyst-independent functions are still considered "undrug-resistant" targets. Furthermore, a large number of oncogeneic proteins, such as transcription factors, chromatin regulators, and low-molecular-weight GTPases, are difficult to target directly pharmaceutically. PROTACs are designed to target the target protein (usually an oncogeneic protein) for degradation by hijacking the endogenous E3 ligase and ubiquitin-proteasome system. An example of an oral PROTAC drug is ARV-110, which targets androgen receptors for degradation and recently received FDA approval for a Phase I clinical trial in 2019 for the treatment of patients with metastatic castration-resistant prostate cancer.

[0255]

[0301] In some embodiments, molecule 110 contains a zinc finger nuclease (ZFN) or a transcription activator-like effector nuclease (TALEN). Mechanistically, a pair of ZFN monomers must bind to DNA, typically by associating with a DNA strand of opposite polarity in a head-to-head configuration. This catalyzes a double-strand break (DSB) in DNA. TALEN consists of a DNA-binding domain composed of modular TALE repeats fused with a FokI nuclease domain. Each TALE repeat consists of 33-35 amino acids and recognizes a single nucleotide. Specificity is determined by two hypervariable residues known as Repeated Variable Diresidues (RVDs). In practice, TALE repeats can be assembled in a fairly straightforward manner to pair with a desired DNA sequence nucleotide by nucleotide. Similar to ZFNs, a pair of TALEN monomers is required to introduce a DSB. ZFNs prefer G-rich sequences, while TALENs typically bind to low-G content sites that strictly begin with T bases. Therefore, the range of DNA sequences that can be targeted by both ZFNs and TALENs is limited.

[0256]

[0302] The CRISPR / Cas9 system is more flexible because it only requires designing and synthesizing sgRNA complementary to the target sequence, and it is generally understood that targeting is easier and faster. Multiple sequences can be targeted simultaneously, and protein optimization is not required. Due to these characteristics, in some cases, CRISPR / Cas technology is preferred over ZFN and TALEN.

[0257] Distribution in the body

[0303] An important characteristic of molecular transfer within the body is its distribution within the body. For molecules to successfully transfer into cells, they must be present at the desired location and concentration within the body at the desired time, in conjunction with the delivery of energy for the transfer.

[0258]

[0304] In some embodiments, this is achieved using a dedicated infusion method. In some cases, it will be understood that the infusion method is designed to deliver molecules 110 at a considerably higher concentration than that typically used in virus-based gene vectors or in vitro laboratory techniques to the vicinity of target cells. This can be achieved in various ways. In some cases, target cells are injected with a solution containing a high concentration of molecules. For example, in some embodiments, the solution contains 1 to 1000 times, e.g., 100 to 500 times, the amount of molecules used in virus-based gene vectors or in vitro laboratory techniques. In some embodiments, 1 to 5 mg / ml of molecules is used. This is significantly different from the more common concentration of 0.5 to 2 mg / ml. In other embodiments, a considerably larger volume of solution is delivered to the vicinity of the target cells. This allows for an increase in the amount of molecules at the target site without increasing the concentration of the delivered solution. For example, 5 to 50 times, e.g., 10 to 25 times, the volume of solution used in virus-based gene vectors or in vitro laboratory techniques may be used. In some embodiments, 0.5 to 5 ml of molecules is used. This differs significantly from the more common volume of 1-2 ml. In some embodiments, it will be understood that both increasing concentration and increasing volume may be used. In some embodiments, increasing the volume of solution in the vicinity of target cells is achieved by delivering the solution to multiple locations around or near the target cells. In some cases, this is achieved by using an energy delivery device 102 having multiple tines, as illustrated in Figure 6. In such embodiments, the solution may be delivered to various locations through the individual tines so as to surround the target tissue region. For example, these locations may be 0.5 mm to 5 cm, particularly 1 to 25 mm, and more specifically 5 to 20 mm from the target tissue region. In some embodiments, it will be understood that different types of solutions, such as solutions of different concentrations, solutions of different volumes, and / or solutions containing different types of molecules, may be delivered through different tines.

[0259]

[0305] In some embodiments, molecule 110 or other components of the solution are optimized to enhance their availability for transfer to target cells within the body. For example, in some embodiments, the molecule is modified to improve its solubility and therefore distribution. In some embodiments, these molecules include linearized DNA, c3 DNA, or supercoiled DNA that can aid in transfer to cells. In other embodiments, the DNA is conjugated with targeted adducts, such as those that are attracted toward a specific target cell population (e.g., toward an antigen / protein on a cell). Normally, naked plasmid DNA undergoes minimal cellular uptake due to its high negative net charge, very large molecular size, and susceptibility to enzymatic degradation. However, conjugation and similar methods can mitigate this. In some cases, lipid and polymer nanoparticles utilize cation groups to embed and condense DNA plasmids for protection from nuclease degradation and neutralization of the negative charge. Similarly, by using gold nanoparticles, DNA plasmids can be protected from nuclease activity through a PAMAM (polyamidoamine) polymer conjugated to the gold surface, which creates a negative phosphate backbone and charge interaction. In some embodiments, modified nucleotides are incorporated into specific sites on the plasmid to create specific conjugation points that can be used for nanoparticle attachment. These nanoparticles then serve as a platform for numerous interactions of various polymers, specifically targeting aptamers for cellular uptake. For example, a plasmid / nanoparticle complex conjugated with a CD44-aptamer facilitates targeted delivery of the plasmid to breast cancer cells that highly express CD44 on their cell membranes. Various aptamers can be used to direct the plasmid to specific cell types.

[0260]

[0306] In some embodiments, genetic material or other components are combined with additives that increase their ability to remain within a target tissue region. For example, in some embodiments, genetic material such as DNA is combined with a polymer such as polyethylene glycol that assists the passage of the genetic material into cells. In other embodiments, genetic material or other components are combined with a solution that improves its solubility or its ability to spread within tissue volume, such as a solution that modifies the surface tension of a fluid, such as a surfactant, glycol, dimethyl sulfoxide, lipofectamine, or cationic analgesic.

[0261]

[0307] In some embodiments, the in vivo distribution of molecule 110 is improved by inducing extravasation of fluid within local areas of the body. Devices, systems, and methods for inducing such extravasation may involve the delivery of energy, such as a specific type of pulsed electric field energy or other preferred energy types. Such extravasation is typically from nearby vascular structures, lymphatic vessels, or other tissues receiving energy. In some cases, extravasation is edema or edema-like, where fluid leaks from capillaries into surrounding tissue. Edema occurs when an abnormal volume of fluid accumulates in tissue, either intracellularly (cellular edema) or within a collagen-mucopolysaccharide matrix distributed in the interstitium (interstitial edema). The methods for extravasation described herein focus on extracellular matrix swelling or interstitial edema. Spontaneous interstitial edema can result from abnormal changes in pressure (hydrostatic pressure and colloid osmotic pressure) acting across microvessel walls, changes in the molecular structure that constitutes the barrier to fluid and solute flux of the endothelial wall, manifesting as changes in the permeability coefficient and the osmotic reflectance coefficient of plasma proteins, or changes in the lymphatic outflow system. However, the methods described herein induce edema or extravasation by the delivery of specific energy.

[0262]

[0308] In some cases, extravasation of fluid from blood vessels transports molecules delivered intravenously to the target tissue area. In other cases, fluid leaks from blood vessels, and molecules are delivered locally or regionally to the target tissue site by injection or other means. In yet other cases, extravasation is used alone without molecular delivery. Examples of improvements in treatment include, but are not limited to, conditioning the target tissue, increasing the availability of molecules, increasing the uniformity of molecular availability, increasing naturally limited access to the target tissue, expanding the treatment area, and reducing potential undesirable side effects.

[0263]

[0309] Figures 11A to 11C illustrate various stages of an embodiment of an extravasation procedure. In this embodiment, the energy delivery device 102 comprises an elongated shaft 106 and an energy delivery body 108 positioned near the distal end of the elongated shaft 106. In this embodiment, the energy delivery body 108 consists of a single electrode, and its distal tip 101 is configured to penetrate tissue in or near a portion of tissue in the target tissue region T. In other embodiments, it will be understood that the energy delivery body 108 has a non-invasive tip and is delivered via a separate instrument capable of penetrating tissue. As illustrated in Figure 11A, the energy delivery body 108 is positioned within the target tissue region T near a vascular BV, such as a capillary. In this embodiment, the molecule 110 is delivered to the target tissue region T through the vascular BV, such as by IV administration. Such a molecule 110 is specific to the procedure, such as providing genetic material for gene transfer.

[0264]

[0310] Figure 11A shows that only a small amount of molecules 110 penetrated the target tissue region, while a considerable amount remained within the vascular BV. Then, as illustrated in Figure 11B, at least one dose of conditioning energy is delivered from the energy delivery body 108 to the target tissue region, as indicated by the dashed line 113. Typically, the conditioning energy includes a dedicated form of PEF energy, but it will be understood that other types of dedicated energy may be used to induce desired extravasation. In this embodiment, dedicated PEF energy reversibly disrupts the functional integrity of the fluid barrier of endothelial cells within the vascular BV by affecting the permeability coefficient and the osmotic repulsion coefficient of plasma proteins, among other things. This disruption makes it more difficult for the barrier to restrict the movement of fluids and macromolecules from the blood to the interstitial tissue of the surrounding tissue. This causes the fluid and solutes containing molecules 110 from the vascular BV to extravasate and spill into the target tissue region, as illustrated in Figure 11C. The PEF energy typically disrupts capillaries while minimizing cellular damage in the targeted region.

[0265]

[0311] This extravasation process can occur over a period of time, ranging from 5 seconds to 30 seconds to 15 minutes. Therefore, in some cases, it is desirable to initiate the delivery of molecule 110 to the vascular structure before delivering conditioning PEF energy to ensure the highest concentration and availability of molecule 110 in the bloodstream. The duration of extravasation and edema development may vary depending on various factors, including the targeted organ, the parameters used, and the specific objectives of the treatment. For example, molecule 110 not delivered at high systemic concentrations may require the maximum extravasation effect before the treatment procedure. Similarly, highly bioavailable molecule 110 may require less extravasation effect before the treatment procedure. Typically, it is desirable that the vascular BV is most leaky during the period when the concentration of molecule 110 passing through these vascular BVs is highest, and therefore, the extravasation of molecule 110 into the interstitial environment of the targeted tissue region is maximized.

[0266]

[0312] Inducing extravasation offers several advantages. These include, but are not limited to, creating a larger treatment area, conditioning the treatment area to better accept therapeutic procedures, increasing molecular availability, improving the uniformity of molecular availability, increasing molecular delivery to naturally restricted locations (e.g., delivery across the blood-brain barrier), and reducing the potential for side effects of the procedure.

[0267]

[0313] In this embodiment, molecule 110 is intended to be taken up by cells in the targeted treatment area. In some embodiments, induced extravasation alone is sufficient to increase the uptake of molecule 110 by cells in the target tissue area. In other embodiments, the uptake of molecule 110 is further facilitated by the delivery of therapeutic energy, as will be described in more detail herein. In some embodiments, it will be understood that the therapeutic energy consists of PEF energy having a different waveform from the conditioning PEF energy. In some embodiments, it will be understood that the therapeutic PEF energy is delivered using the same energy delivery device 108 positioned within the target tissue area. In other embodiments, the therapeutic energy is delivered using a different device.

[0268]

[0314] In some embodiments, the conditioning PEF energy has a waveform that includes monophase, long-duration (greater than 500 μs) pulses. Figure 12A illustrates an example of such a PEF energy waveform provided by the energy delivery algorithm 152 of a generator 104 used to induce extravasation. In this embodiment, the waveform consists of a series of pulses 400, each having a pulse width 402 and amplitude (determined by a set voltage 404), with each pulse 400 separated by a delay 406. In this embodiment, this pulse width is considered long-duration, exceeding 500 microseconds. In this embodiment, the delay 406 between pulses 400 is in the range of 10 μs to 10 s, e.g., 1 ms, 500 ms, 1 second, 2 seconds, 5 seconds. Although two pulses 400 are illustrated in Figure 12A, conditioning may be achieved with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pulses or more than 10 pulses. In some embodiments, this PEF energy is not designed to induce uptake of molecule 110 by cells within a target tissue region, and therefore a set of pulse parameters (e.g., voltage, frequency, inter-pulse delay, etc.) can be utilized. However, in some embodiments, it will be understood that the treatment energy itself may be the same as that in Figure 12A. In some embodiments, the waveform is biphasic, as illustrated in Figure 12B, where each pulse 400 is biphasic and has a pulse width 402 separated by a delay 406. In this embodiment, the pulse width 402 is again considered long, exceeding 500 microseconds. In this embodiment, the delay 406 between pulses 400 is in the range of 1 μs to 1 second, for example, some examples being 1 μs to 10 μs, 10 μs, 1 μs to 100 μs, 100 μs, 1 μs to 250 μs, 250 μs, 1 μs to 500 μs, 500 μs, 1 ms, 2 ms, 5 ms, or 1 to 5 ms. In some embodiments, it will be understood that the pulses 400 are polarized such that some pulses 400 have a positive amplitude and some pulses 400 have a negative amplitude.Such polarity reversals may be symmetrical or asymmetrical. In some embodiments, it may also be understood that pulses 400 are grouped by polarity. Any preferred number of pulses may be present in each group, and each group may have the same number of pulses or different numbers of pulses. For example, there may be six positive pulses followed by two negative pulses, or four positive pulses followed by one negative pulse. Thus, various combinations are possible. Such grouping may be symmetrical or asymmetrical.

[0269]

[0315] In some embodiments, it will be understood that conditioning energy is delivered to a target tissue region to increase its cellular resistance to eventual cell death. Cells subjected to sublethal stress are known to undergo repair and prophylactic responses to stress, substantially developing resistance to subsequent stresses of similar or different natures and enhancing their resilience. For example, in some embodiments, conditioning energy causes the release of heat shock proteins (HSPs). HSPs are a family of proteins produced by cells in response to exposure to stress conditions, such as conditioning energy as described herein. While HSPs were initially described in relation to heat shock, they are now known to be expressed in other stresses, including low temperatures and UV light exposure, as well as during wound healing or tissue remodeling. Many members of this group exert chaperone function by stabilizing new proteins to ensure correct folding, or by assisting in the refolding of proteins damaged by cellular stress. This increased expression is transcriptionally regulated. The dramatic upregulation of heat shock proteins is a crucial part of the heat shock response and is primarily induced by heat shock factors (HSFs).

[0270]

[0316] In some embodiments, preheating of tissue or cells (prior to the delivery of molecule 110) may initiate the expression of heat shock proteins that play a role in cell injury, repair, and survival. In such embodiments, a warm solution, such as warm saline, may be injected into the treatment site, where molecule 110 is delivered along with energy delivery after a waiting period. The waiting period may be several minutes, several hours, or several days after the delivery of the warm solution. In some embodiments, the waiting period is 5 to 30 minutes, 1 to 2 hours, or 1 to 2 days.

[0271]

[0317] In other embodiments, tissue or cells are heated using energy delivery device 108. In such embodiments, energy is delivered at a controlled rate to maintain the local temperature within a specific range, for example, 40-50°C for treatments of less than 10 minutes. In some embodiments, it will be understood that heat shock proteins are triggered at approximately 41°C. Therefore, by using sublethal pulsed electric field delivery, upregulation of heat shock proteins and other damage repair preparations can be promoted before stronger treatment pulsed electric fields. This increases the resilience of cells to injury from pulsed electric fields and improves their ability to deliver molecules to a significant number of cells without causing unwanted excessive cell death.

[0272]

[0318] Therefore, in one embodiment, conditioning energy is delivered to the target treatment area, raising the temperature in at least a portion of the treatment area to, for example, 45°C. This induces extravasation of fluid into that area. A drug, gene, or other type of molecule is delivered by injection and benefits from the advantages of extravasation as described herein. A treatment, such as therapeutic PEF energy, is then delivered to the target treatment area. Since the cells in the treatment area are pre-conditioned to resist cell death, a larger number of cells survive this treatment protocol. This is beneficial for gene therapy or other types of treatment that rely on cell survival.

[0273]

[0319] Further examples of devices, methods, and energy waveforms providing such preconditioning (e.g., leading to extravasation) are provided in U.S. Provisional Patent No. 63 / 209,335, filed June 10, 2021, entitled "INDUCED EXTRAVASATION BY ENERGY DELIVERY TO TISSUE" (incorporated herein by reference for any purpose).

[0274]

[0320] In some embodiments, the in vivo distribution of the molecule is optimized by a controlled combination approach to the delivery of molecule 110 to the body. In some embodiments, delivery of molecule 110 by IV combined with local injection provides a greater synergistic effect for enhanced transfer than IV alone or local injection alone.

[0275]

[0321] Example 1: Delivery of plasmid DNA encoding highly sensitive green fluorescent protein (EGFP) to the posterior gastrocnemius muscle of balb / c mice using PEF energy. Plasmid DNA was administered to mice in three ways: 1) systemic injection (in the tail vein) of 20 μg of plasmid (equivalent to 1 mg / kg) in 200 μl of saline; 2) local injection of 200 μl of saline containing 200 μg of plasmid DNA into the gastrocnemius muscle using an electropharmaceutical needle; and 3) a combination of the above administration routes, with systemic injection first, followed by local administration before / simultaneously with energy delivery. Two different PEF energy delivery algorithms were used: 1) PEF algorithm A (a biphasic shredding cycle as described in a later section), and 2) PEF algorithm B (alternating DC as described in a later section). Depending on the energy delivery algorithm used, local delivery of plasmid DNA was performed one minute before PEF algorithm B (8-second protocol) or simultaneously with PEF algorithm A (5-minute protocol) to ensure slow and consistent material delivery while accumulating energy in the tissue. To achieve consistent and accurate delivery of genetic material over 5 minutes, 200 μl was delivered at a rate of 0.04 ml / min using an infusion pump. Mice were maintained under gas anesthesia with isoflurane during the delivery of genetic material and energy. After the procedure, the mice were recovered, kept alive for 3 days, euthanized, and the gastrocnemius muscle was collected. The muscle tissue was weighed, mechanically cut with a blade, dissolved in RIPA buffer, and homogenized by sonication. After ultracentrifugation, protein was extracted from the homogenized tissue and used to measure EGFP protein by ELISA. The results were normalized to the weight of each tissue to obtain the pg / mg concentration of EGFP protein in the sample. The results are illustrated in Figure 13. It should be understood that IV+IM = local delivery following intravenous delivery. Five samples were measured for PEF algorithm A, and nine samples for PEF algorithm B. It should be understood that IM = local intramuscular delivery. Five samples were measured for PEF algorithm A, and ten samples were measured for PEF algorithm B.It should be understood that IV stands for intravenous systemic delivery. Four samples were measured for PEF algorithm A and four samples for PEF algorithm B. Figure 14 shows the average results from Figure 13. Thus, in this example, IV alone did not produce a measurable transfer, but the IV+IM protocol produced a larger transfer than would have been expected from the combination of IV alone and IM alone. Furthermore, in this example, PEF algorithm B using the IV+IM protocol produced the largest transfer level.

[0276]

[0322] It is understood that gene transfer is usually maximized when plasmid DNA is added before the application of an electrical pulse that enables entry into the cell membrane and induces electrophoresis of DNA in contact with the cell membrane. Another hypothetical mechanism of gene electrotransfer is that during electric field delivery, negatively charged DNA molecules move due to electrophoretic force, making more contact with the cell membrane than free diffusion would. Large plasmid DNA molecules form aggregates on the cell membrane during the electrical pulse and enter the cell via endocytosis. For this additional reason, when plasmids are administered systemically, it is desirable to have high concentrations of the plasmid at more difficult migration sites.

[0277] Energy

[0323] As previously stated, the energy delivery device 102 delivers the energy provided by the waveform generator 104 according to at least one unique energy delivery algorithm 152. Various energy delivery algorithms 152 specifically designed for intracellular transfer procedures are provided. In some embodiments, the algorithm 152 defines a signal having a waveform that includes a series of pulses with delays in between. In such embodiments, the algorithm 152 specifies signal parameters such as energy amplitude (e.g., voltage), pulse width, delay period, and number of pulses, to give some examples. It will be understood that the energy delivery algorithm 152 generates a waveform that maximizes the transfer but avoids adverse effects, particularly those specific to intracellular transfers.

[0278]

[0324] Conventional exovivotive techniques for electrogene transfer typically require energy from waveforms with pulses containing very long DC portions (e.g., 1–100 ms, e.g., 2–50 ms). Delivering long DC pulses of a certain polarity, or a series of pulses all of the same polarity, increases the risk of ablation and the resulting size of ablated lesions. This is usually due to electrolysis, pH imbalance, and net charge accumulation in the tissue. The current flowing through biological material produces various effects, including electrolysis, ion topolesis, and electroosmotic flow, in addition to migration and potential Joule heating. These can contribute to the formation of ablations.

[0279]

[0325] Furthermore, when these unbalanced waveforms are used in the body, various effects can occur within the body. These unbalanced waveforms can activate motor neurons and skeletal muscles, thereby causing injury and pain in the patient, and adversely affecting treatment outcomes. Consequently, the energy delivery algorithm 152 described herein is configured to avoid these adverse effects by avoiding these imbalances.

[0280]

[0326] Figure 15A illustrates an example of a waveform provided by the energy delivery algorithm 152. In this embodiment, the waveform consists of a series of pulses 400, each having a pulse width 402 and amplitude (determined by a set voltage 404), with each pulse 400 separated by a delay 406. In this embodiment, three pulses 400 are illustrated, but the transition may be achieved with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 pulses or more than 10 pulses. In some embodiments, energy is delivered in such a unipolar form, and the amplitude or set voltage 404 of each pulse is, to give some examples, 1-500V, 1-250V, 1-100V, 10-100V, 10-70V, 10-50V, 10-40V, 10-30V, 10-20V, 10V, 20V, 30V, 40V, 50V, 60V, 70V, 80V, 90V, and 100V. The voltage used and considered may be the peak of a square waveform, the peak of a sinusoidal or sawtooth waveform, or the RMS voltage of a sinusoidal or sawtooth waveform.

[0281]

[0327] In some embodiments, the pulse width is, to give some examples, 1 ms, 2 ms, 5 ms, 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, 100 ms, 1-20 ms, 1 ms-50 ms, 1 ms-100 ms, 2 ms-100 ms, and 1-2 ms. In some embodiments, the delay between pulses is, to give some examples, 0.01-5 seconds, 0.01-0.1 seconds, 0.01-0.5 seconds, 0.01-1 second, 0.5 seconds, 0.5-1 second, 1 second, 1-1.5 seconds, 1-2 seconds, 0.5-2 seconds, 2 seconds, and 1-3 seconds. In some embodiments, the number of pulses is, to give some examples, 1 pulse, 2 pulses, 3 pulses, 4 pulses, 5 pulses, 6 pulses, 7 pulses, 8 pulses, 9 pulses, 10 pulses, and more than 10 pulses.

[0282]

[0328] In a preferred embodiment, it will be understood that the pulses 400 are polarized such that some pulses 400 have positive amplitude and some pulses 400 have negative amplitude. Such reversal reduces the charge imbalance which can lead to increased ablation and / or muscle contraction as described above. In some embodiments, it will be understood that the polarity alternates in the form of up, down, up, down. For example, Figure 15B illustrates the alternating DC waveforms provided by algorithm 152. Here, a positive pulse 400' is followed by a negative pulse 400'', and this is repeated in an alternating pattern. Again, each pulse 400', 400'' has a pulse width 402 and amplitude (determined by the set voltage 404), and each pulse 400', 400'' is separated by a delay 406. It will be understood that these parameters may differ, such as each pulse having different pulse widths 402, amplitudes 404, and delays 405. However, it is desirable that the positive on-time and negative on-time balance out over time. In some embodiments, the amplitude 404 is in the range of 10 to 500V, such as 50 to 100V. In some embodiments, The pulse duration 402 is in the range of 0.5 to 200 ms, for example, 1 to 100 ms, 2 to 50 ms, or 20 to 30 ms. In some embodiments, the inter-pulse delay 406 is in the range of 10 ms to 10 s, for example, 200 ms to 3 s, or 500 ms to 2 s. If necessary, the inter-pulse delay 406 may be synchronized with the patient's ECG. In some embodiments, the procedure includes 1 to 100 pulses, for example, 1 to 20 pulses, or 1 to 10 pulses. These can result in a total procedure time of 0.5 to 500 ms (typically 20 to 200 ms). Table 1 below provides examples of various parameter combinations for achieving the transition of molecule 110.

[0283] [Table 1]

[0284]

[0329] It can be seen that algorithm B, shown in Figures 13 and 14, generated a waveform with the shape of Figure 15B. In the experiment, algorithm B resulted in a higher level of migration under both conditions of intramuscular injection alone and in combination with intravenous injection. Therefore, this waveform demonstrates that significant migration of affected cells is preserved while reducing or eliminating the potential lethal effect of PEF.

[0285]

[0330] In some embodiments, it will also be understood that the pulses 400 may be grouped by polarity. For example, Figure 15C illustrates an example of a waveform provided by the energy delivery algorithm 152, in which two pulses 400' have positive polarity followed by two pulses 400'' have negative polarity. It will be understood that any suitable number of pulses may be present in each group, and each group may have the same number of pulses or different numbers of pulses. For example, there may be six positive pulses followed by two negative pulses, or four positive pulses followed by one negative pulse. Thus, various combinations are possible. Such grouping may be symmetrical or asymmetrical.

[0286]

[0331] In some embodiments, one or more pulses 400 are “chopped,” divided, or segmented into a series of segment pulses of the same phase. As a result, each pulse 400 becomes longer due to the inclusion of delays between segment pulses, which can be considered segment delays. Figure 16 illustrates an embodiment of a waveform based on a conventional single-phase waveform (and therefore considered a “basic” waveform with a basic pulse), but has been significantly modified to optimize transition in an in vivo environment. The single-phase waveform used in conventional gene therapy is a DC pulse, typically having a pulse width of 1 to 50 ms, usually 20 ms. Here, a pulse similar to the single-phase waveform (i.e., positive pulse 400') is shown, but here, the positive pulse 400' is chopped or divided into multiple segment pulses 401 with segment delays 405 in between. Thus, in this embodiment, the positive pulse 400' has a pulse width in the range of 10 to 100,000 microseconds, which is considerably longer than the single-phase DC pulse used in conventional gene therapy. However, the "on time" of pulse 400' is the same as in the basic waveform because, by segmentation, a delay is introduced rather than an additional pulse. Figure 16 also shows a negative pulse 400'' separated from the positive pulse 400' by delay 406. In this embodiment, the negative pulse 400'' is similar to the positive pulse 400' but has negative polarity. Thus, the waveform in Figure 16 is significantly different overall from the conventional single-phase waveform. To avoid muscle activation, segmented pulses break down the long driving force for the molecule to migrate into the cell. Electrophoresis will be understood to be a crucial element in driving the molecule 110 toward and into the cell as it migrates into the cell. Typically, the timescale driving electrophoretic motion in molecules of the size and molecular weight of molecule 110 described herein is larger than the timescale at which these membrane potential differences induce muscle activation. For example, neuronal activation may occur with 10 ms of PEF energy delivery, and skeletal muscle activation may occur with 40 ms of PEF energy delivery.Such activation is avoided by "cutting" the long driving force (pulse 402) into a series of short / short-duration pulses 401 having pulse widths 403. This allows molecules 110 to be gradually delivered into the cell, and the interruption (interval delay 405) allows for the relaxation of the membrane potential load, thereby preventing muscle excitation. Therefore, small interruptions do not weaken the driving force of molecules 110 into the cell, but rather weaken muscle excitation.

[0287]

[0332] In some embodiments, pulses 400', 400'' are relatively long, for example, 10 to 100,000 μs or 100 to 50,000 μs, and it will be understood that they have a delay 406 in the range of 100 μs to 10 s, for example, 1 ms to 5 s or 50 ms to 2 s. These pulses 400', 400'' consist of multiple interval pulses 401 (for example, to give a few examples, The pulses may be divided into interval pulses 401 of up to 10,000, 10-2,000, or 20-1,000, where the duration of each interval pulse is 0.05-50 μs, for example, 0.5-20 μs or 1-5 μs, and the interval delay is 1-100,000 μs, for example, 1-10,000 μs or 10-2,000 μs. In some embodiments, the on-time does not exceed 10 μs, which can be considered an inflection point for the initiation of muscle stimulation. In such embodiments, a delay of 10 ms between pulses 400' and 400'' is desired. Therefore, the pulses 400' and 400'' and interval pulses 401 in Figure 16 are for illustrative purposes only to highlight the components of the waveform, and in many embodiments they are not drawn to a constant scale because it is extremely difficult to illustrate the individual thousands of interval pulses. The pulses 400', 400" paired with 409 shown in Figure 16 can be considered cycles, and it will be understood that they are typically repeated 1 to 1000 times, especially 1 to 200 times, more specifically 1 to 100 times, and more specifically 40 to 50 times, for example. Various repetition counts are acceptable, as long as they are not so small that the muscle contractions become unified (E > 250 ms) and not so large that the effects do not accumulate (E < 10 s).

[0288]

[0333] In some embodiments, the voltage amplitude will be understood to be in the range of 10 to 250V, for example, 50 to 250V or 50 to 100V.

[0289]

[0334] Table 2 below provides examples of various parameter combinations for achieving molecular transfer.

[0290] [Table 2]

[0291]

[0335] Figure 17 illustrates an embodiment of a waveform based on a biphasic AC waveform. Here, two pulses, a positive pulse 400' and a negative pulse 400'', are shown separated by a delay 406, and these two pulses form one cycle. However, here, the positive pulse 400' is chopped into multiple segment pulses 401 with segment delays 405 in between. Similarly, the negative pulse 400'' is also chopped into multiple segment pulses 401 with segment delays 405 in between. Similar to the embodiment in Figure 16, these segment pulses subdivide the long driving force as molecules move into the cell to avoid muscle activation. Again, small interruptions do not dull the driving force of molecules 110 into the cell, but they do dull muscle excitation. Generally, these embodiments change polarity more frequently than the embodiment in Figure 16. Similarly, in these embodiments, cycles 409 are typically grouped into packets (with inter-packet delays in between), and one or more packets are delivered during treatment.

[0292]

[0336] In some embodiments, the pulse width 402 is somewhat shorter than the pulse width in Figure 16, for example, 0.1 to 10,000 μs, 10 to 1,000 μs, or 5 to 500 μs, but has a similar delay 406 in the range of 100 μs to 10 s, for example, 1 ms to 5 s or 50 ms to 2 s. These pulses 402 may be divided into multiple interval pulses 401 (for example, to some examples, up to 1,000 interval pulses 401, 5 to 100 interval pulses 401, or 10 to 50 interval pulses 401), where the duration 403 of the interval pulses is 0.05 to 50 μs, for example, to some examples, 0.5 to 20 μs or 1 to 5 μs, and the interval delay 405 is 1 to 100,000 μs, for example, 1 to 10,000 μs or 10 to 2,000 μs. Similarly, the pulses in Figure 17 are for illustrative purposes only to highlight the components of the waveform, and in many embodiments they are not drawn to a constant scale because it would be extremely difficult to illustrate the individual thousands of interval pulses. It will be understood that the cycle 409 illustrated in Figure 17 is typically repeated 1 to 1,000 times, particularly 1 to 100 times, and more specifically 1 to 10 times, for example, to some examples. The number of repetitions can vary, as long as it is not so small that muscle contractions become unified (E>250ms) and not so large that the effect does not accumulate (E<10s). In some embodiments, cycles are grouped into packets with inter-packet delays. In some embodiments, packets include 1 to 100 cycles, e.g., 1 to 50 cycles, 1 to 20 cycles, 1 to 10 cycles, 1 to 5 cycles, etc. In some embodiments, multiple packets are delivered during a single treatment or during a single transfer of molecule 110 to target cells. In some embodiments, 1 to 100 packets are delivered, e.g., 1 to 50 packets, 1 to 20 packets, 1 to 10 packets, 1 to 5 packets, etc.

[0293]

[0337] In some embodiments, the voltage amplitude is understood to be in the range of 10 to 5000V, for example, 10 to 1000V, 10 to 50V, 50 to 100V, 50 to 200V, 10V, 50V, 100V, 200V, 500V, and 1000V.

[0294]

[0338] Table 3 below provides examples of various parameter combinations for achieving molecular transfer.

[0295] [Table 3]

[0296]

[0339] It will be understood that Algorithm A, shown in Figures 13 and 14, generated a waveform with the shape shown in Figure 17. In the experiment, Algorithm A resulted in a significant level of migration under both conditions of intramuscular injection alone and in combination with intravenous injection. Therefore, this waveform demonstrates that significant migration of affected cells is maintained while reducing or eliminating the potential lethal effect of PEF.

[0297]

[0340] As stated, pulse 400 can have various characteristics such as various amplitudes (determined by the set voltage 404) and pulse widths 402. For example, Figure 18A illustrates an example of a waveform provided by the energy delivery algorithm 152, in which a first pulse 400' having a first voltage 404' and a first pulse width 402' is followed by a different second pulse 400'' having a second voltage 404'' and a second pulse width 402''. Here, the first voltage 404' is higher than the second voltage 404'', and the first pulse width 402' is narrower than the second pulse width 402''. Thus, the first pulse 400' can be considered high and short in relation to the second pulse 400'', which can be considered low and long. In this embodiment, these pulses 400', 400'' are then repeated. In some embodiments, short, high pulses have a voltage amplitude 404' in the range of 100 to 1000 V and a pulse width 402' in the range of 50 ns to 1 ms, for example, 50 ns to 100 μs or 50 ns to 10 μs. Low, long pulses have a voltage amplitude 404'' in the range of 5 V to 100 V and a pulse width 402'' in the range of 1 ms to 50 ms.

[0298]

[0341] In some embodiments, such waveforms are considered multifunctional waveforms. For example, in some embodiments, short high-voltage pulses create conditions that enable migration, and long low-voltage pulses deliver molecules into the cell by electrophoresis or the like. In some cases, molecules 110 that approach the cell, driven by electrophoresis or simply by rotation, vibration, or shaking, "attach" to the cell and are drawn into the cell when the cell performs membrane repair. Thus, some molecules 110 may enter the cell by endocytosis during repair or other cellular states. In some embodiments, the short high-voltage pulses are monophasic or biphasic. Also, in some embodiments, the long low voltage is DC or alternating DC.

[0299]

[0342] Figure 18B illustrates a similar embodiment, where the second pulse 400'' is negative. Therefore, the first voltage 404'' is greater than the second voltage 404'' and has the opposite polarity. This balances the charge. In some embodiments, it is desirable to balance the amplitude and on-time of the positive pulse with the amplitude and on-time of the negative pulse. This can be visualized by making the pulse areas equal. For example, if the area of ​​the positive pulse 400'' is equal to the area of ​​the negative pulse 400'', then those pulses are considered balanced. This allows for pulses of various shapes. Such balancing reduces the net charge in either the positive or negative direction, thereby reducing muscle stimulation.

[0300]

[0343] Figure 18C illustrates another similar embodiment, where pulses 400', 400'' have the same polarity within a group or set, but the next pulse set has the opposite polarity. It will also be understood that these sets may be separated by a delay, which is typically in the range of 1 to 3 seconds and may be synchronized with the patient's heart rate. It will also be understood that transitions may be achieved by delivering one or more sets. In some embodiments, the delivery of additional sets may be predetermined, triggered (by one or more sensors, for example), or applied at the user's discretion.

[0301]

[0344] In some embodiments, the first pulse 400' induces rotation of at least a portion of the molecule 110, such as pure rotation without translational effects. In some cases, this is achieved with the first pulse 400' having a pulse width 402' of 50 ns to 1 ms, e.g., 50 ns to 100 μs or 50 ns to 10 μs, such that it has a native frequency of 1 GHz or higher. In such embodiments, the second pulse 400'' has a pulse width 402'' of 1 ms to 50 ms, such that it has a native frequency of less than 1 GHz, such as in the range of 1 kHz to 999 MHz. The second pulse 400'' induces either a combined translational and rotational effect or a pure translational effect. This combination of pulses 400', 400'' accelerates the migration of the molecule 110 to the cell toward migration (e.g., the rotational effect at the start of treatment is utilized to boost the later translational effect). It will be understood that multiple first pulses 400' and / or second pulses 400'' may be used in combination to adjust their effects.

[0302]

[0345] As previously mentioned, in some embodiments, a series of pulses 400 are delivered in units of packets, where packets are separated by inter-packet delays. Figure 19 illustrates a packet 411 containing a series of pulses 400, in this embodiment, the packet 411 consists of four pulses. Each pulse has a voltage 404 and a pulse width 402. In this embodiment, each pulse is separated by a delay 406, and similarly, each packet is separated by an inter-packet delay 415. It will be understood that the packet 411 may consist of a series of identical pulses or of different pulses. Similarly, the inter-pulse delays 415 may be regular or irregular. Figure 20 illustrates a packet 411 containing a series of pulses 400 having alternating polarities, which can be considered a series of biphasic pulses, each biphasic pulse having a switch-time delay 407. Here, each packet 411 consists of four pulses with alternating polarities, i.e., two biphasic pulses.

[0303]

[0346] In each embodiment described herein, it will be understood that pulses (whether monophase pulses, biphase pulses, alternating DC pulses, etc.) can be grouped into packets 411. In some embodiments, the packet delay is up to 20 seconds, for example, 0.1-20s, 0.1-20s, 10s, 0.1-5s, 0.1-2s, 0.1-1s, 0.5-1s, 0.5-5s, 0.1s, 0.5s, 1s, 5s, 10s, etc. In some embodiments, the inter-packet delay is synchronized with the ECG.

[0304]

[0347] In some embodiments, as illustrated in Figures 21 and 22, a series of high-voltage, high-frequency pulses are followed by a series of low-voltage, low-frequency pulses, and this combination assists in the transfer of molecule 110 to target cells. For example, as illustrated in Figure 21, a first set of pulses 420 is delivered, which includes multiple high-voltage, high-frequency pulses, optionally in packets. Such pulses may prepare the cells for subsequent transfer.

[0305]

[0348] Examples of such high-voltage, high-frequency pulses are provided, to name a few, in U.S. Patent No. 10,702,337 entitled “Methods, apparatuses, and systems for the treatment of pulmonary disorders” and PCT / US2020 / 028844 entitled “DEVICES, SYSTEMS AND METHODS FOR THE TREATMENT OF ABNORMAL TISSUE” (as incorporated herein by reference). Such pulses 420 may be used for ablation in other clinical applications, but in the case of migration or molecules 110, such pulses are adapted to prepare cells for migration. These pulses 420 are typically biphasic (having a positive pulse and a negative pulse, which combine to form one cycle), and the cycles are grouped into packets. The number of cycles is half the number of pulses in each biphasic packet. In some embodiments, the number of cycles is set between 1 and 100 per packet (including all values ​​and subranges within that range). In some embodiments, the number of cycles is up to 5 cycles, up to 10 cycles, up to 25 cycles, up to 40 cycles, up to 60 cycles, up to 80 cycles, up to 100 cycles, up to 1,000 cycles, or up to 2,000 cycles (including all values ​​and subranges in between). Packet duration is determined by the number of cycles, among other factors. Generally, a higher number of cycles results in a longer packet duration and a greater amount of energy delivered. In some embodiments, the packet duration is in the range of approximately 50 to 1,000 microseconds, for example, 50 μs, 60 μs, 70 μs, 80 μs, 90 μs, 100 μs, 125 μs, 150 μs, 175 μs, 200 μs, 250 μs, 100 to 250 μs, 150 to 250 μs, 200 to 250 μs, and 500 to 1,000 μs. In other embodiments, the packet duration is in the range of approximately 100 to 1000 microseconds, for example, 150 μs, 200 μs, 250 μs, 500 μs, or 1000 μs.

[0306]

[0349] Typically, there is a default pause time between packets. However, there may be algorithms to vary the pause time systematically, or there may be no pause time applied between packets at all. Table 4 provides examples of parameter values ​​for such pulse 420.

[0307] [Table 4]

[0308]

[0350] In some embodiments, a first pulse set 420 is followed by a delay 422 (e.g., 100 microseconds to 2 seconds), followed by a second pulse set 424. In this embodiment, the second pulse set 424 consists of multiple low-voltage, low-frequency pulses. Figure 21 illustrates that the first pulse 426 of the second pulse set 424, which lasts up to 10 microseconds, is followed by a delay 406 (e.g., up to 1 ms), followed by a second pulse 428. In this embodiment, since the second pulse 428 has the opposite polarity to the first pulse 426, the delay 406 can be considered a switch-time delay 407. In some embodiments, it will be understood that there is no delay 406 / 407 between pulses 426 and 428. In some cases, the first pulse set 420 prepares the cells for molecular migration (e.g., makes the cells more receptive to molecules or migration processes), and the first pulse set 420 initiates the migration process. Next, a second pulse set 424 assists in the transfer of molecules into cells (e.g., delivering or pushing molecules into cells). If necessary, these pulse sets 420, 424 may be repeated as a pattern.

[0309]

[0351] Figure 22 illustrates another example of a waveform having diverse segments, where a first pulse set 420 is delivered, which includes multiple high-voltage high-frequency pulses, as may be in units of packets (e.g., as described above in relation to Figure 21). Again, examples of such high-voltage high-frequency pulses are provided, to name a few, in U.S. Patent No. 10,702,337 entitled “Methods, apparatuses, and systems for the treatment of pulmonary disorders” and PCT / US2020 / 028844 entitled “DEVICES, SYSTEMS AND METHODS FOR THE TREATMENT OF ABNORMAL TISSUE” (as incorporated herein by reference), as also described above herein. In some embodiments, the first pulse set 420 is followed by a delay 422 (e.g., 100 microseconds to 2 seconds), followed by a second pulse set 424. In this embodiment, the second pulse set 424 consists of a plurality of low-voltage, low-frequency pulses. In this embodiment, the second pulse set 424 consists of a series of biphasic pulses without a switch-time delay, and the second pulse set 424 lasts for approximately 100 microseconds to 5 milliseconds. Again, in some cases, the first pulse set 420 prepares the cells for molecular migration or initiates the migration process. The second pulse set 424 then assists in the migration of molecules into the cells (e.g., delivering or pushing molecules into the cells). If necessary, these pulse sets 420, 424 may be repeated as a pattern.

[0310]

[0352] It will be understood that these pulses 400 may not be square waves, but sine waves, or may have other shapes. For example, Figure 23 illustrates another embodiment of the pulse waveform, where each pulse is composed of multiple increasing and decreasing voltages, forming a pyramidal shape.

[0311]

[0353] In any of the embodiments described herein, it will be understood that the set voltage 404 may vary depending on whether the energy is delivered in a unipolar or bipolar manner. In bipolar delivery, a lower voltage can be used due to the smaller and more directional electric field. The bipolar voltage selected for use in treatment depends on the gap distance between the electrodes, whereas a unipolar electrode arrangement using one or more distant dispersed pad electrodes can be delivered without much consideration for the precise placement of the catheter electrode and the dispersed electrode placed on the body. In embodiments of unipolar electrodes, the dispersed electrode may consist of a pad or any other receiving electrode. Typically, a dispersed electrode functions as such due to its size (large enough not to have a local effect on the place of installation) and / or its placement (at a distance that avoids local effects and does not pose a risk of electric arc discharge). However, in some embodiments, the dispersed electrode is small and may have some effect on its place of installation, but such effects may be benign and incidental. For example, the molecules being delivered may not be close enough to the dispersed electrode to avoid migration, or the migration in that area may be insignificant. In unipolar electrode configurations, due to the dispersion behavior of energy delivered through the body, larger voltages are typically used to reach the dispersed electrodes with an effective gap distance of approximately 10 cm to 100 cm. Conversely, in bipolar electrode configurations, the relatively close electrode activity regions of approximately 0.5 mm to 10 cm, including 1 mm to 1 cm, have a greater impact on the electrical energy concentration and the effective amount delivered to the tissue from that gap distance.

[0312]

[0354] Energy delivery can be activated by various mechanisms, such as the use of a button on the energy delivery device 102 or a foot switch operably connected to the generator 104. Such activation typically provides a single amount of energy, defined by the number of pulses delivered and the voltage of those pulses.

[0313]

[0355] Figures 24A to 24C illustrate additional embodiments of the waveform provided by algorithm 152. Here, a high-frequency, fast oscillation is added to the pulse to provide an additional push or rotation to the molecule 110 while the pulse as a whole drives the molecule 110 toward the cell. This may be particularly beneficial when a large molecule 110 or aggregate of molecule 110 has contact with the cell membrane but has difficulty entering the cell. Such a surge in energy may provide the push required to enter the cell. Referring to Figure 24A, in this embodiment, a first pulse 400' is provided having a first pulse width 402 (e.g., 20 ms), followed by a second pulse 400'' having a second pulse width 402 (e.g., 20 ms). Here, pulses 400', 400'' are separated by a delay 406, such as a delay of 5000 μs. In this embodiment, each of the pulses 400' and 400'' is positive and has a set voltage or primary voltage V1. In this embodiment, the voltage oscillates around the primary voltage V1, generating a spike 413. Such oscillations typically occur at up to 25% of the primary voltage (e.g., 0.1-25% or 1-25%), and more typically at up to 10% of the primary voltage (e.g., 0.1-10% or 1-10%, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15%, 20%). This is % or 25%. For example, in the case of 10% oscillation, a pulse 400' with a primary voltage V1 of 50V may oscillate between 55V and 45V, generating a 55V spike 413. Similarly, a pulse 400' with a primary voltage V1 of 1000V may oscillate between 1100V and 900V by 10% oscillation. Each spike 413 provides an additional push or rotation to the molecule 110 during translation. Thus, the spikes 413 resulting from the oscillation cause pulses 400', 400'' to appear as a crown. It will be understood that the pulse may contain spikes 413 throughout the entire pulse length (i.e., pulse width 402), as illustrated, or that spikes 413 may occur at any point along the pulse length.Similarly, any number of spikes 413 may be present (to some examples, including 100–10,000 spikes, 100–5,000 spikes, 500–5,000 spikes, and 500–1,000 spikes). In some embodiments, the oscillation is an oscillation at a frequency above 1 GHz that causes rotation of at least some of the molecules 110, such as pure rotation without translational effects. This combination of spikes 413 within pulses 400', 400'' assists in the translocation of molecules 110 to target cells (e.g., increasing the push to or into the cells).

[0314]

[0356] Figure 24B illustrates an embodiment similar to that of Figure 24A, where each of the pulses 400' and 400'' is in the negative direction. Similarly, Figure 24C illustrates pulses 400' and 400'' in the reverse direction. It will be understood that the waveforms described and illustrated herein are merely examples to illustrate various waveform concepts and are not intended to be a comprehensive list of all possible waveforms. Similarly, these waveform concepts are presented as a series of examples along with the characteristics of specific waveforms that may vary. Thus, it will be understood that the waveform embodiments, characteristics, and concepts may be used in various combinations and are not limited to the combinations described herein. For example, the concept of a “chopped” waveform may be applied to any of the fundamental waveforms, where the pulses of that fundamental waveform are chopped as described herein. Similarly, the concept of a crown-like oscillation may be applied to any of the fundamental waveforms, where the pulses of that fundamental waveform are modified to include oscillation. Furthermore, waveforms may be combined in various combinations, for example, where a part of one waveform embodiment may be followed by a part of another waveform embodiment. Similarly, waveforms that induce extravasation may be used in combination with any of the waveforms disclosed herein or variations thereof.

[0315] enhancement

[0357] The ability to deliver molecule 110 to tissues or cells can be modified using various enhancements. For example, in some embodiments, an adjuvant material is introduced into the body, such as by being added to a solution that carries the molecule, where the adjuvant material makes cells more receptive to the transport of small or macromolecules. Examples of adjuvant materials include macromolecular nanoparticles, liposomes, PEGylated liposomes, lipofectamine, cell-permeable peptides (CPCs), dimethyl sulfoxide (DMSO), cholesterol, or other materials known to interact with the fluidity and mechanics of the cell membrane. In some embodiments, the adjuvant material is injected, and the injection pressure is selected or adjusted to enhance the uptake of molecule 110 by cells.

[0316]

[0358] In other embodiments, tissues or cells are heated or cooled to alter their ability to successfully receive the molecule 110. For example, in some embodiments, cells are heated or cooled, such as by heating or cooling the molecule-carrying solution 110, to induce improved transfer efficiency or improved cell viability after energy delivery. In some embodiments, heating cells may increase membrane fluidity and thus increase the acceptance of molecule 110. In other embodiments, cooling cells may increase rigidity and increase the likelihood of forming “cracks” that increase the acceptance of molecule 110.

[0317]

[0359] In other embodiments, preheating of tissue or cells (prior to the delivery of molecule 110) may initiate the expression of heat shock proteins that play a role in cell injury, repair, and survival. In such embodiments, a warm solution, such as warm saline, may be injected into the treatment site, where molecule 110 is delivered along with energy delivery after a waiting period. The waiting period may be several minutes, several hours, or several days after the delivery of the warm solution. In some embodiments, the waiting period is 5 to 30 minutes, 1 to 2 hours, or 1 to 2 days.

[0318]

[0360] In some embodiments, tissue or cells are heated by the use of energy delivery device 108. In such embodiments, energy is delivered at a controlled rate to maintain the local temperature within a specific range (e.g., 40-50°C for treatments of less than 10 minutes). In some embodiments, it will be understood that heat shock proteins are triggered at approximately 41°C. Thus, by using sublethal pulsed electric field delivery, upregulation of heat shock proteins and other damage repair preparations can be promoted before stronger treatment pulsed electric fields. This increases the resilience of cells to injury by pulsed electric fields and improves their ability to deliver molecules to a significant number of cells without causing unwanted excessive cell death.

[0319]

[0361] As previously mentioned, among other functions, the processor 154 modifies and / or switches the energy delivery algorithm, monitors energy delivery and any sensor data, and reacts to the monitored data via a feedback loop. In some embodiments, the processor 154 is configured to execute one or more algorithms for running a feedback control loop based on one or more measured system parameters, one or more measured tissue parameters and / or a combination thereof. In some embodiments, the parameters include temperature, which can be maintained within a specific range by controlling the cadence of energy delivery. This may be useful for enhancing cellular uptake, immune response, overall safety, etc.

[0320]

[0362] It will be understood that enhancements may be applied before, during, or after the delivery of molecule 110, and / or before, during, or after the delivery of treatment energy. In some embodiments, the adjuvant material is administered to the patient at a desired interval between multifunctional waveforms (e.g., between a high, short pulse and a low, long pulse of an asymmetrical waveform). This can assist in the delivery or intrusion of the adjuvant material into the cells.

[0321]

[0363] In some embodiments, it will be understood that local tonicity is adjusted by delivering isotonic or hypertonic saline to the treatment site. sensor

[0322]

[0364] As previously mentioned, in some embodiments, the energy delivery device 102 comprises one or more sensors that can be used to measure pressure, temperature, impedance, resistance, capacitance, conductivity, pH, optical properties (coherence, echo brightness, fluorescence), dielectric constant or photodielectric constant and / or conductance, to name a few. In some embodiments, one or more of their electrodes act as one or more sensors. In other embodiments, one or more sensors are separate from the electrodes. The sensor data can be used to plan treatment, monitor treatment, and / or provide direct feedback via the processor 154, after which the energy delivery algorithm 152 can be modified.

[0323] A. Pressure sensing

[0365] It is understood that cells generally respond to mechanical stimuli. One such type of mechanical stimulus is pressure. Generally, hydrostatic pressure is determined by the volume of interstitial fluid and the general compliance of the targeted interstitial tissue. It is important to note that this varies by tissue type, as some organs are encased in more rigid / less flexible structures (e.g., brain, kidneys), while others expand / contract more freely (e.g., lungs, muscles, skin). While increased hydrostatic pressure can increase the permeability of local cell membranes, excessive interstitial pressure can certainly lead to tissue damage and cell death. Mammalian cells have been shown to undergo various forms of apoptosis at pressures of 200–300 MPa and succumb to more immediate cell death at pressures of 300–400 MPa.

[0324]

[0366] Therefore, it is often beneficial to pay attention to the pressure the target cells are enduring, especially during the injection of molecules 110, where the local injection pressure can affect the target cells. As a result, in some embodiments, the energy delivery device 102 typically includes a pressure sensor positioned to monitor the local injection pressure.

[0325]

[0367] In some embodiments, feedback from a pressure sensor is used to prevent reaching harmful pressures. For example, pressure readings from the sensor may be displayed to the user, one or more algorithms 152 may be modified based on the readings, and / or a warning or shutdown may occur when the pressure reaches a predetermined threshold.

[0326]

[0368] However, hydrostatic pressure plays a crucial role in cell membrane permeabilization. In some cases, a desirable increase in extracellular pressure facilitates the migration of molecules 110, such as exogenous DNA, into the cell. Furthermore, hydrostatic pressure often plays a significant role in the intratissue distribution of injected molecules 110. Therefore, in some embodiments, pressure measurements (e.g., external pressure measurements taken at the proximal end of device 102) can be used by the user as a measure to ensure optimized migration conditions. In such embodiments, the pressure can be increased or decreased to fall within a desired predetermined pressure range. In some embodiments, the pressure is measured (minimum or zero injection flow rate) before injecting molecules 110 to determine the basal intracellular pressure (Pi=P1|Q1=0). Considering the optimal defined target pressure difference (Pd=Pi-Pe) for achieving migration, and knowing the hydrostatic pressure of the system relative to the solution (Rapplicator), the pressure is set to the desired pressure difference (P1=Pi+Pd+Q1 * Adjust the inflow rate (Q1) until the Rapplicator (R) is increased. By optimizing the infusion rate and the start of treatment application in this way, the desired results can be obtained.

[0327]

[0369] When the delivery method includes induced extravasation, as described above, pressure sensor measurements can be used to monitor edema levels before, during, and / or after the injection of molecules 110 and treatment energy. Thus, by adjusting the induction of extravasation, a desired edema level can be achieved at specific points in time throughout the treatment protocol.

[0328]

[0370] Various pressure sensors 200 may be used. In some embodiments, the pressure sensor 200 is positioned along the distal tip of the energy delivery device 102, such as as shown in Figure 25A. Here, the energy delivery device 102 comprises a needle-shaped energy delivery body 108, which is at least partially covered by an insulating sleeve 202. As shown, the molecule 110 travels through the energy delivery body 108 to reach a nearby target tissue region. This allows the pressure sensor 200 to monitor the pressure during the injection of the molecule 110 and the application of treatment energy. In other embodiments, the pressure sensor 200 is positioned along the distal end of the energy delivery device 102, but proximal to the tip, as shown in Figure 25B. Here, the pressure sensor 200 is positioned along an insulating sleeve 202 that at least partially covers the energy delivery body 108. By obtaining relative pressure measurements at the tissue level, the user can understand the tissue distribution of the injected molecule 110. Considering the solution injected at known pressures and flow rates (values ​​easily measurable near the proximal end of device 102), additional relative measurements at the distal end provide information not only for understanding the pressure profile over time but also for understanding the spatial distribution of molecules along the target tissue.

[0329]

[0371] In some embodiments, the pressure sensor 200 includes a strain gauge transducer. The strain gauge transducer is typically characterized by exhibiting a change in output mode depending on the quantity being measured (i.e., strain, electrical resistance, or wavelength). Sensitivity is determined by the relative change in resistance with respect to length.

[0330]

[0372] In other embodiments, the pressure sensor 200 includes a diaphragm displacement sensor. The diaphragm displacement sensor is based on micro-electromechanical system technology, where the sensor has a bendable plane (diaphragm) on a sealed cavity. The diaphragm bends or deforms in response to changes in pressure. The resulting output form may be capacitance-based or piezoelectric transducer-based. In some embodiments, the sensor 200 is located at the distal tip of the energy delivery device 102, while its corresponding diaphragm is located proximal to the sensor 200, thus enabling measurement of pressure drop over the distance between them. If the placement of such a sensor is difficult due to size constraints or other reasons, a pressure-sensing optical fiber may be preferred.

[0331]

[0373] In some embodiments, the energy delivery device 102 includes an expandable member 204 positioned along its distal end, as illustrated in Figure 25C. Here, the expandable member 204 is mounted on an insulating sleeve 202 that at least partially covers a needle-shaped energy delivery body 108. Such positioning of the expandable member 204 can help prevent backflow of the solution of molecules 110 back into the path formed by inserting the energy delivery device 102. This can improve the pressure distribution within the target tissue, allowing the molecules 110 to be migrated more effectively. The expandable member 204 can also prevent movement of the needle-shaped energy delivery body 108 during PEF energy delivery. In some embodiments, a pressure sensor 200 mounted on the expandable member 204 monitors the pressure within the expandable member 204. This ensures proper expansion of the expandable member 204. Furthermore, in some embodiments, the pressure sensor 200 monitors tissue pressure during the infusion of molecules 110 and / or during PEF energy delivery.

[0332] B.pH sensing

[0374] It will be understood that cells generally respond to biological stimuli. One biological stimulus that plays an essential role in cell survival is pH. Assuming pH is stable, relatively small changes within that pH range can potentially affect virtually all cellular processes, including metabolism, membrane potential, cell growth, transmembrane transport, cytoskeletal polymerization, and muscle cell contractility.

[0333]

[0375] In some embodiments, a pH sensor is provided to monitor local pH (e.g., located along the energy delivery device 102, such as near the distal tip). By using the pH measurement, the user may be warned or one or more algorithms 152 may be modified to avoid reaching harmful acidity / alkalinity, but more importantly, it may be optimized and used as a measure for optimal transition conditions / settings.

[0334] C. Temperature sensing

[0376] Another biological stimulus that plays an essential role in cell survival is temperature. Relatively small temperature changes can potentially affect virtually all cellular processes, including metabolism, membrane potential, cell growth, transmembrane transport, cytoskeletal polymerization, and muscle cell contractility. In some embodiments, a temperature sensor is positioned along the energy delivery device 102 to monitor local temperature. This makes it possible to monitor and maintain local temperature within a normal physiological range while simultaneously reducing or eliminating thermal damage during energy delivery. Furthermore, in some embodiments, temperature sensing is used in combination with moderate cooling / heating related to the delivery of molecules 110 to a target site, ultimately improving the transition as described earlier. It will be understood that various types of temperature sensors, such as thermocouples, resistance thermometers, and optical fibers, may be used.

[0335] Transition and Ablation

[0377] In some embodiments, it will be understood that the devices, systems, and methods described herein may be adapted to provide ablation in addition to the transfer of molecule 110. This may be particularly useful when treating tumors in the body. In such examples, the energy delivery device 10 is configured to access the tumor, for example, percutaneously or intraluminally. The generator 104 comprises one or more algorithms 152 for delivering PEF energy having a waveform that provides ablation at a specific location in the target tissue and a waveform that provides the transfer of the molecule to other locations in the target tissue. In some embodiments, the PEF energy creates various treatment zones extending radially outward from the energy delivery body 108. For example, the zone closest to the energy delivery body 108 (i.e., the central zone) is resistant to immediate cell death via necrosis, etc., while the zone surrounding the central zone (i.e., the peripheral zone) receives the transfer of molecule 110 for gene therapy, etc.

[0336]

[0378] It will be understood that some of the PEF energy waveforms described above in this specification can be adapted to provide ablation in addition to the migration of molecule 110. For example, in some embodiments, the alternating DC waveform illustrated in Figure 15B is configured to provide ablation in addition to the migration of molecule 110 by modifying various parameter values. Table 5 below provides examples of various parameter combinations for achieving ablation in the central zone and migration of molecule 110 in the peripheral zone.

[0337]

[0379] [Table 5]

[0338]

[0380] In some embodiments, the alternating polarity "cut" waveform of Figure 16 is configured to provide ablation in addition to the migration of molecule 110 by modifying various parameter values. Table 6 below provides examples of various parameter combinations used with this waveform to achieve ablation in the central zone and migration of molecule 110 in the peripheral zone.

[0339]

[0381] [Table 6]

[0340]

[0382] In some embodiments, the alternating polarity "cut" biphasic waveform of Figure 17 is configured to provide ablation in addition to the migration of molecule 110 by modifying various parameter values. Table 7 below provides examples of various parameter combinations used with this waveform to achieve ablation in the central zone and migration of molecule 110 in the peripheral zone.

[0341]

[0383] [Table 7]

[0342]

[0384] As previously mentioned, in some embodiments, the energy delivery device 102 comprises a shaft 106 having an energy delivery body 108 near its distal end, the energy delivery body 108 comprising a plurality of tines 600. Similarly, referring to Figure 6, in some embodiments, a first section 106a acts as an energy delivery body 108, and one or more tines 600 also act as energy delivery bodies 108. Each of the different energy delivery bodies 108 may deliver the same type of energy or different types of energy, and similarly, the energy delivery bodies 108 may act in groups. In particular, in some embodiments, the first section 106a delivers energy for tissue excision, and one or more tines 600 deliver energy for the transfer of molecules to the cells of the tissue. In other embodiments, it will be understood that certain tines deliver energy for tissue excision, and other tines deliver energy for the transfer of molecules to the cells of the tissue. In some embodiments, it will be understood that the same tine may be used to deliver different types of energy at different time points. Similarly, in some embodiments, it will be understood that the same tine may be used to deliver molecule 110 or energy at different time points. And in some embodiments, the same tine may be used to deliver molecule 110 and energy simultaneously. These embodiments may be particularly useful when treating unwanted tissue such as tumors, where the tumor is excised to ensure complete removal, and molecule 110 is delivered around it.

[0343] Further clinical applications A. Delivery to cardiac tissue

[0385] Figure 26 illustrates a method for delivering molecule 110 to cells in the heart H using an energy delivery device 102. In this embodiment, the energy delivery device 102 comprises an elongated shaft 106 and an energy delivery body 108 positioned near its distal end. Furthermore, the shaft 106 comprises one or more delivery ports 702 located at a distance proximal to the energy delivery body 108 that delivers molecule 110. In some embodiments, one or more delivery ports 702 are positioned in the aorta A above the aortic valve AV when the energy delivery body 108 is positioned in the left ventricular LV. In the embodiment of Figure 26, one or more delivery ports 702 are positioned above the aortic valve AV and near the coronary bifurcation, and the energy delivery body 108 is positioned near the wall of the left ventricular LV. This allows molecule 110 to be delivered to the coronary bifurcation, and the molecule 110 can enter the coronary circulation. Figure 27 illustrates the coronary artery CA and the location of the coronary artery bifurcation relative to the aorta A and aortic valve AV. By delivering molecule 110 to the aorta A above the aortic valve AV, molecule 110 can enter the coronary circulation to reach various tissues within the heart H. Because the aortic valve AV is a one-way valve, molecule 110 is largely prevented from entering the left ventricle LV. Energy deliverers 108 located within the heart H, such as in the left ventricle LV, can deliver energy to the wall of the heart H so that molecule 110 circulating nearby through the coronary artery CA can penetrate the tissue cells of the heart wall H.

[0344]

[0386] It will be understood that the energy delivery device 102 may have various configurations to deliver energy to the heart H at one or more locations simultaneously or in a certain pattern. Similarly, additional delivery devices may be used to deliver energy and / or molecules 110 to various locations within or near the heart H. This may be useful when delivering energy to the most easily accessible part of the heart H from various chambers.

[0345]

[0387] In some embodiments, it will be understood that molecule 110 can be delivered from the distal end of the energy delivery device 102, for example, within the left ventricular LV, so as to be located near the wall to which energy is delivered, or it can be delivered from the distal end of the energy delivery device 102 through penetration into the wall so that molecule 110 is delivered into the wall before or during energy delivery. It will be understood that any combination of molecule 110 and energy delivery may be used. In any of these scenarios, molecule 110 is delivered to the tissue so that cells can take up molecule 110 during energy delivery.

[0346] B. Delivery to extramedullary hematopoietic tumors

[0388] Hematopoiesis is the process of producing all types of blood cells, including their formation, development, and differentiation. Prenatally, hematopoiesis occurs in the yolk sac, then the liver, and finally the bone marrow. However, in some patients, hematopoiesis occurs spontaneously or pathologically outside the bone marrow (e.g., in chronic anemia such as thalassemia, sickle cell disease, and myeloproliferative disorders; in patients with classic myeloproliferative disorders, particularly chronic idiopathic myelofibrosis; and in other myeloproliferative disorders with polycythemia vera and, rarely, essential thrombocythemia, which are considered "secondary myelofibrosis"). Less frequently, it can also be seen in patients with myelodysplastic / myeloproliferative disorders (e.g., chronic myelomonocytic leukemia) or myelodysplastic syndromes. This is considered extramedullary hematopoiesis (EMH), referring to hematopoiesis that occurs outside the bone marrow. Extramedullary hematopoiesis is most commonly observed in the spleen and liver, but can also occur in other areas (e.g., lymph nodes, lungs, serosal surfaces, chest, adrenal glands, genitourinary system, skin, and retroperitoneal and paravertebral spaces).

[0347]

[0389] EMH is hypothesized to be a compensatory response to an increased need to produce blood. Ineffective red blood cell production in patients with abnormal hemoglobinopathy can lead to the formation of extramedullary hematopoietic (benign) tumors or lumps in various parts of the body. Such lumps can reach dimensions of several centimeters. Since such lumps are generally benign, they are rarely removed. In some embodiments, cells in such lumps, such as CD34+ cells (hematopoietic stem cells, HSCs), become targets for the transfer of molecules 110 using PEF energy, devices, and methods described herein. Such locations, particularly within the liver or spleen, are relatively accessible, and immune cells produced after procedures such as gene mutation editing can be released into the body to cure or alleviate the corresponding disease. C. Delivery to bone marrow

[0348]

[0390] In some embodiments, the transfer of molecule 110 to stem cells is carried out directly in the patient's bone marrow using PEF energy, devices, and methods described herein. In some embodiments, the energy delivery device 102 is configured to access the bone marrow itself, and in other embodiments, the energy delivery device 102 is configured to advance into the bone marrow after an accompanying device has provided access. In some embodiments, hematopoietic stem cells are the target of the transfer. In such embodiments, the iliac crest (in the pelvis) is perforated to access the target cells in the bone marrow. The iliac crest is bone rich in hematopoietic stem cells, is easily accessible, and is routinely used for accessing bone marrow for bone marrow donation. The perforation creates a hole through which the energy delivery device 102 can advance to deliver PEF energy. As stated, molecule 110 may be delivered through the energy delivery device 102 or through a variety of other routes. Finally, molecule 110 transfers to the target cells with the help of PEF energy. Thus, genetic blood disorders are treated by directly (in vivo) delivering molecule 110, such as gene editing material, to hematopoietic stem cells (HSCs) in the bone marrow.

[0349]

[0391] Direct delivery of molecule 110 to the bone marrow is superior to the exovivo method. The exovivo method isolates hematopoietic stem cells (CD34+ cells) from mobilized peripheral blood. Mobilized peripheral blood is systemic circulating blood treated with a mobilizer. The term "mobilization" refers to the process that encourages stem cells to migrate from the bone marrow into the peripheral circulation, where they can be recovered via leukocyte extrusion. The mobilizer mobilizes a large number of hematopoietic stem cells and progenitor cells from the bone marrow and extravasates them into the bloodstream. Due to the nature of these active ingredients, as well as the complexities surrounding donor recruitment and collection, donors must be closely monitored and collections continuously managed to ensure both donor safety and successful collection.

[0350]

[0392] Furthermore, when stem cells are amplified in vitro using growth factors, these stem cells can sometimes transform into cancer cells. In fact, in clinical trials of CRISPR, some sickle cell disease patients developed blood cancer after reinfusion of cells. Reinjecting edited stem cells into patients requires multiple rounds of chemotherapy to suppress the immune system and make them suitable for transplantation; otherwise, the body will reject those cells.

[0351]

[0393] In the bone marrow, only 5-10% of CD34+ cells are HSCs. Exovivo CD34+ selection cannot completely eliminate T cells, and this affects allogeneic dysmorphic transplantation, as graft-versus-host disease (GvHD)-free transplantation requires pure HSCs without T cell contamination. Approximately 95% of endogenous HSCs are in the G0 phase of the cell cycle. Following the recruitment of classical cyclophosphamide / granulocyte colony-stimulating factor (G-CSF), all bone marrow HSCs enter the cell cycle, expand, and upregulate the expression of CD47, the macrophage's "don't eat me" signal. Bone marrow HSCs enter the bloodstream in G0 / G1, home to the bone marrow via the interaction of HSC integrin α4β1 with sinusoidal endothelial cell adhesion molecules, cross the macrophage domain, and home to the CXCL12 niche via the HSC CXCR4 receptor. When HSCs are cultured in vitro with factors, they enter the cell cycle, move into G1, and enter the S phase in about 30 hours. When HSCs in the S / G2 / M phase are injected intravenously, these HSCs lose their ability to home to the bone marrow. After 2-3 days of culture, the majority of HSCs become progenitor cells that cannot self-replicate. Thus, only a small number of CD34+ cells are HSCs to begin with, and the homing of HSCs that are in the cell cycle is insufficient.

[0352]

[0394] Furthermore, the biological activity and environment of HSCs influence the genetic modification of HSCs by viral vectors. Unlike cells, viral vectors cannot home from the blood to the bone marrow and cannot penetrate the bone marrow blood vessels to enter the HSC niche. With current recruitment methods, both the vector and G0 / G1 HSCs are present in the blood only for short periods, and even the recruited HSCs are a sparse population. While the viral envelope protein can promote fusion with HSCs, allowing the viral vector to release cargo into cells, vectors for gene therapy also require HSC-specific binding sites and off-target delivery, and integration can still be problematic. Nevertheless, if lentiviral vectors with an envelope external domain that binds to HSC enrichment markers are created, in vivo gene transfer may become possible, and experimental manipulation of recruitment regimens may increase the residence time of HSCs in the blood, improving vector accessibility. None of these strategies intend to deliver molecule 110 and its subsequent effects (e.g., gene editing) directly in the bone marrow as described herein.

[0353]

[0395] The CRISPR / Cas9 system will be understood as a preferred system for gene editing. The CRISPR / Cas9 system can be used for gene editing at various locations in the body, including bone marrow, using the PEF energy, devices, and methods described herein. This is in contrast to conventional tools for gene editing in HSCs (e.g., lentiviral vectors expressing both Cas9 and guide RNA, which are not optimal because they have low titers, sustained Cas9 expression after transduction, and make transduction difficult to achieve). Similarly, conventional electroporation using Cas9 + guide RNA ribonuclear particles, while efficient in vitro, can be highly toxic and is therefore unsuitable for in vivo application.

[0354] D. Delivery to mesenchymal stem cells

[0396] In some embodiments, the transfer of molecule 110 to mesenchymal stem cells is carried out directly within the body, such as in the patient's bone marrow or adipose tissue, using the PEF energy, devices, and methods described herein. Mesenchymal stem cells (MSCs) are a desirable therapeutic target for many diseases, particularly those related to the musculoskeletal system. MSCs are adult stem cells that can differentiate into mesodermal cells, including osteoblasts, chondrocytes, and adipocytes. MSCs are rare non-hematopoietic progenitor cells that were first isolated from bone marrow (BM). While primarily found in BM and adipose tissue, there have also been cases of isolation from peripheral blood, umbilical cord blood, synovial membrane, deciduous teeth, amniotic fluid, and perivascular regions.

[0355]

[0397] MSCs are both low immunogenic and immunomodulatory, can home to damaged tissue, and have been found to depend on the secretion of bioactive molecules to initiate healing in the repair process. MSCs express a variety of chemokine receptors that enable migration in response to chemokine-attracting gradients produced by inflammatory injury sites. Therefore, genetic modification of these cells (e.g., by using the devices and methods described herein) could be used to treat a wide range of diseases, from cancer to cardiovascular or skeletal disorders.

[0356]

[0398] In vivo transfer of molecule 110 to MSCs overcomes many of the drawbacks of ex vivo transfer. First, prolonged cell culture is avoided, eliminating the resulting effect of increased heterogeneity of the cell population. Increased diversity in the cell population makes it difficult to clearly define the functional populations and mechanisms involved in detectable effects. Furthermore, prolonged culture can lead to the downregulation of functional surface proteins such as CXCR4, and cells grown ex vivo for extended periods exhibit reduced migration and homing abilities in vivo. Thus, any problems regarding the transfer of results from bench to bedside are avoided. Finally, in addition to concerns about serum-dependent growth when transplanted into humans, sarcoma formation from transplanted MSCs, as seen in mice, is common, but cultured MSCs can acquire mutations and transform. This, too, is avoided with in vivo transfer.

[0357]

[0399] It is desirable to transfer molecule 110 to MSCs in the microenvironment where MSCs are present. The microenvironment in which MSCs are present, whether it is perivascular, BM, or another region, substantially affects the homing, differentiation, and regenerative capabilities of MSCs. The surrounding cellular and extracellular environment is also thought to affect surface proteins and epitopes that can be used as identification markers.

[0358]

[0400] It will be understood that MSCs may be directly targeted in vivo for gene editing or gene therapy. These manipulations of MSCs are for the following diseases:

[0359]

[0401] This improves the engraftment of hematopoietic stem cell transplants. MSCs were first isolated from the bone marrow (BM), a crucial site for hematopoiesis, and are now thought to play a vital role in hematopoiesis. Hematopoietic stem cells (HSCs) require stromal support cells for proper differentiation and to maintain quiescence within the endothelial niche of the BM; both of these support functions can be performed by osteoblasts, which are descendants of MSCs. This has provided the rationale for using MSCs in conjunction with HSC transplantation, with the expectation of promoting the engraftment and proliferation of donor HSCs. Genetically modified MSCs present at the transplant site may improve engraftment.

[0360]

[0402] Hypophosphatasia: Hypophosphatasia is a disease characterized by reduced bone mineralization due to tissue-nonspecific alkaline phosphatase (ALP) inactivating mutations in osteoblasts and chondrocytes. Differentiation of transplanted MSCs into osteoblasts may be an effective measure to deliver a cell population capable of producing normal ALP enzymes. Whole BM transplantation in combination with cultured osteoblasts is used to treat infants suffering from hypophosphatasia. However, local (intra-BM) transfection of ALP enzymes into MSCs using the devices and methods described herein will improve outcomes.

[0361]

[0403] Osteogenesis Imperfecta: Osteogenesis imperfecta (OI) is a group of at least nine genetic disorders characterized by incomplete bone elongation and a high risk of fracture, with varying degrees of severity. In most affected individuals, it is caused by a deficiency of type I collagen, i.e., abnormal synthesis. The primary treatment involves bone resorption-inhibiting bisphosphonates to increase bone density, but this does not address the underlying cause. MSCs can be useful in treating patients with OI because they can differentiate into osteoblasts and supply normal collagen I. MSCs with successful targeted deletion of mutant collagen process and form collagen and collagen fibrils similar to those of wild-type cells. In vivo, these cells are capable of ectopic osteogenesis in mice.

[0362]

[0404] Cardiovascular Diseases: Over the past decade, research on MSCs and cardiovascular diseases has been steadily increasing. Specifically, in myocardial infarction, the plasticity, homing, and inflammatory regulation of MSCs, which begin to act after hypoxia-induced damage to cardiomyocytes due to arterial occlusion, are all important properties. Since the cardiomyocytes lack the ability to efficiently replace these cells, the goal of any treatment is to replace the cardiomyocytes. The use of genetically modified MSCs attempts to improve the homing, survival, and paracrine-mediated effects of MSCs. In some embodiments, the devices and methods described herein for delivering molecule 110 to MSCs in the bone marrow are used to overexpress therapeutic genes in MSCs that subsequently migrate to the heart.

[0363]

[0405] Prevention of pulmonary hypertension: Genetically modified MSCs are used to prevent pulmonary ischemia by preventing pulmonary hypertension. The devices and methods described herein may be used for the delivery of molecule 110 to MSCs in the bone marrow.

[0364]

[0406] Cancer: MSCs can preferentially hom to the growth sites of primary and metastatic tumors, allowing for the delivery of antitumor agents to highly specific niches surrounding the tumor. The two main categories of gene targeting are cytotoxic, i.e., pro-apoptotic genes, and immunomodulatory genes. Pro-apoptotic genes: Receptors for TNF-related apoptosis-inducing ligands are expressed on many types of tumors, and when soluble ligands were expressed in MSCs and appropriately localized in xenograft mouse models of human cervical cancer, breast cancer, and glioma, it resulted in reduced growth and tumor size, as well as increased apoptosis and extended survival time. Inducible nitric oxide synthase has also been shown to be a potentially potent antitumor therapy, reducing tumor growth when delivered to a mouse fibrosarcoma model by genetically modified MSCs. A second class of therapeutic transgenes is immunomodulatory targets. MSCs engineered to express IL-2, -7, -12, and -18 reduced tumor size in rodent xenograft models of primary, established, and metastatic tumors. MSCs possessing IFN-α and -β, as well as other immune cytokines such as CX3CL1 / fractalkine, increased apoptosis in tumor cells of prostate, lung, pancreatic, and skin cancers, extending animal survival time through activation of adaptive immune responses by increasing innate immune activity, such as natural killer cells, or T cell activation.

[0365]

[0407] Type 1 diabetes (T1D) is caused by organ-specific autoimmune-mediated loss of insulin-secreting β-cells in the pancreas. People with T1D manage their blood glucose levels with exogenous insulin therapy, but this does not eliminate the development of long-term diabetic complications such as retinopathy, nephropathy, and neuropathy. Currently, pancreatic transplantation or islet transplantation remains the only treatment option, but these procedures are limited by a shortage of donor organs and the need for lifelong immunosuppression. Mesenchymal stem cells (MSCs) are an attractive alternative target cell for autologous and allogeneic treatments of T1D.

[0366]

[0408] Targeting the central nervous system: Due to their homing properties, mesenchymal stem cells (MSCs) can migrate to various parts of the body and act as vehicles to carry out repairs from the venous line to the CNS without eliciting a significant immune response.

[0367]

[0409] In the event of any inconsistency in usage between this document and any document referenced by it, the usage of this document shall prevail.

[0368] Delivery to the eye A. Background

[0410] The retina is a thin layer of tissue that lines the back of the eye. Its purpose is to receive light focused by the lens, convert that light into nerve signals, and send these signals to the brain for visual perception. Therefore, the retina is central to the visual process of converting light into signals that the brain can interpret.

[0369]

[0411] Figure 28 illustrates a cross-section of the retina R near the optic nerve ON. The central region of the retina near the optic nerve ON is called the macula and contains a high density of color-sensitive photoreceptor cells (light-sensing cells). These cells, called cones, produce the sharpest visual images and are responsible for central vision and color vision. The peripheral region of the retina surrounding the macula contains photoreceptor cells called rods, which respond to lower light levels but are not color-sensitive. Rods are responsible for peripheral vision and night vision. Both cones and rods are found within the laminae-coconae (Jakob's membrane) LRC. The optic nerve ON transmits signals originating from the photoreceptors (cones and rods). Each photoreceptor is connected to the optic nerve by small nerve branches. The optic nerve connects to nerve cells that transmit signals to the visual center of the brain, where they are interpreted as visual images.

[0370]

[0412] The optic nerve (ON) and retina (R) are richly supplied with blood and oxygen-carrying vessels. Part of this supply comes from the choroid (CH), a layer of blood vessels located between the retina (R) and the sclera (SC), the outer white layer of the eye. The central retinal artery (the other blood supply to the retina) reaches the retina (R) near the optic nerve (ON) and then branches within the retina (R). Blood is drained from the retina (R) into branches of the central retinal vein. The central retinal vein exits the eye within the optic nerve (ON).

[0371]

[0413] Various diseases and conditions can lead to retinal dysfunction and ultimately blindness. Hereditary retinal degeneration (IRD), also known as hereditary retinal dystrophy, represents a diverse group of progressive visual impairments that can lead to blindness. In IRD, mutations in genes crucial to retinal function lead to progressive photoreceptor cell death and subsequent vision loss. In most people, IRD affects only the eyes. However, some types of IRD are associated with other health problems. IRD is genetically heterogeneous, and to date, more than 260 disease genes have been identified. IRD is the leading cause of blindness in working-age adults. In total, these affect approximately 1 in 4,000 people, or more than 2 million people worldwide.

[0372]

[0414] The development of treatments and therapies that alter or halt the rate of disease progression has been limited to date. Therapies that may slow the degeneration of photoreceptors caused by a range of genetic factors are being studied. Vitamin A and docosahexaenoic acid have been shown to moderately reduce the rate of disease progression in patients with retinitis pigmentosa (RP). Oral valproic acid has been reported to slow visual field progression in a case series of RP patients, but a randomized clinical trial of valproic acid treatment in patients with autosomal dominant RP did not show a significant difference between patients treated with valproic acid and those treated with placebo.

[0373]

[0415] Advances in high-throughput screening are accelerating the identification of cell-targeted and neuroprotective drug candidates. Oxidative damage is involved in photoreceptor degeneration, and preclinical studies of RP have shown that N-acetylcysteine ​​(NAC) and N-acetylcysteinamide (NACA) prevent retinal degeneration. Nonspecific neurotrophic factor therapy with ciliary neurotrophic factor (CNTF) has been shown to slow photoreceptor degeneration in several animal models, but no visual function benefits have been demonstrated in human clinical trials of patients with early or progressive RP.

[0374]

[0416] Furthermore, significant effort is being put into developing gene augmentation therapies for specific genotypes of IRD. Successful clinical trials of gene augmentation therapies for RPE65 and CHM-associated retinal degeneration have been reported. Genome editing, including antisense oligonucleotides, intermittent codon read-through strategies, base editing, and RNA editing, as well as gene-directed pharmacology, may also be promising approaches for genotypes of diseases that may not be suitable for gene augmentation therapy.

[0375]

[0417] However, introducing genetic material into cells presents the challenges mentioned earlier. Unlike many other biological molecules and therapeutic compounds, DNA sequences and nucleic acids are not designed to travel across the cell membrane. Furthermore, they lack endocytosis factors that induce alternative mechanisms for intracellular transport. Therefore, all gene therapies require a combination of at least two features: the encoded therapeutic molecule (DNA sequence, gene, siRNA, etc.) and a route of administration to target cells. One way to deliver genetic material to cells is through the use of viral vectors. The various drawbacks of viral vectors have been discussed earlier.

[0376]

[0418] Electroporation has been known for over 30 years as a method to disrupt the integrity of cell membranes and allow cells in suspension to take up macromolecules. Numerous in vitro studies to test the effects of genes on cell morphology and behavior rely on electroporation as a reliable method of intracellular delivery. Generally, cells are suspended in culture medium pipetted into a cuvette. The cuvette consists of two plate electrodes separated by a predetermined distance. Genetic material is then transfected by delivering a series of pulsed electric fields to the cells. The in vitro environment allows for very precise control of transfection conditions, enabling high levels of gene delivery without killing too many cells. In many cases, protocols specifically optimized for different cell lines have been developed. Overall, in vitro transfection is highly reliable for establishing a fundamental understanding of gene action or for creating transgenic animal models. However, providing viable clinical gene therapy remains challenging. Because cells in culture medium need to be transfected, several additional steps are required before it can be translated into patient treatment. In most cases, gene therapy is performed using an apheresis-based approach. This requires sampling of the patient's cells by blood collection or biopsy. The target cell population is then isolated and enriched. Often, this cell population is amplified to produce transfected cells of a considerable population size. Once the cells are ready, the molecules for gene therapy are electroporated into them. After transfection, the cells are redistributed to the desired site in the patient, such as a target location in the blood or a target organ. These steps add substantial burden, cost, invasiveness, time, and risk to the delivery of gene therapy. Overall, these numerous substantial drawbacks prevent pulsed electric field-based gene therapy from being widely adopted as the most promising treatment option.

[0377] B. Overview

[0419] Devices, systems, and methods are provided for delivering molecules, particularly small molecules and / or macromolecules, to cells within the body, especially target cells inside the eye, and more specifically, target cells inside the retina. Examples of molecules include plasmids, RNA (e.g., messenger RNA (mRNA), small interfering RNA (siRNA), microRNA), antisense oligonucleotides, proteins and / or materials that induce genetic or epigenetic changes in cellular behavior. Such molecules are generally beneficial in the treatment of various diseases and conditions, including, but not limited to, retinitis pigmentosa (RP), rod dystrophy or rod-cone dystrophy, Usher syndrome (USH), Bietti crystalline dystrophy (BCD), Batten disease, Valde-Beetle syndrome (BBS), Alport syndrome, Leber congenital amaurosis (LCA) or early-onset retinal dystrophy (EORD), cone dystrophy, cone-rod dystrophy (CORD), color blindness, congenital stasis nourishment (CSNB), macular dystrophy, Stargardt disease, Best disease, pattern dystrophy, Sosby retinal degeneration, Doin's honeycomb dystrophy, colloideremia, X-linked retinoschisis (XLRS), and other hereditary retinal degenerations (IRDs).

[0378]

[0420] The molecule can be delivered to the eye by various mechanisms, including injection into the vitreous cavity (or the region between the lens and the retina), injection into the suprachoroidal space (the potential space between the sclera and choroid that traverses the periphery of the posterior segment of the eye), and / or injection into a subretinal bleb (a small region formed by separating the retinal pigment epithelium (RPE) that supports the lamina cone (LRC)). The molecule is then delivered to target cells and transmitted through the cell wall of the target cells using a pulsed electric field to exert the desired effect. Thus, in gene therapy, nucleic acids delivered by the molecule are transfected into cells without the use of a virus.

[0379]

[0421] These devices, systems, and methods are superior to known virus-based gene therapies such as adeno-associated virus (AAV) delivery. AAV has limitations in payload capacity. AAV can only accommodate genes smaller than 4.7 kb, and has limited space for gene regulatory regions (e.g., promoters). There are more than 300 disease genes that are too large for AAV-based gene therapy. These include genes that cause many relatively common diseases, such as Stargardt disease, Usher 1B and 1D, and Leber congenital amaurosis-10 (LCA10). In contrast, the nonviral delivery methods described herein can accommodate genes larger than 10 kb and have no space limitations. Therefore, many genes that are excluded from use in AAV delivery can be delivered by the devices, systems, and methods described herein.

[0380]

[0422] The device, system, and method deliver pulsed electric field energy to cells using an energy delivery system 100. Figure 29 illustrates an embodiment of the energy delivery system 100 configured to deliver energy to a portion of an eye. In this embodiment, the energy delivery system 100 comprises a dedicated energy delivery device 102 that can be detachably connected to a waveform generator 104. In this embodiment, the generator 104 comprises a user interface 150, one or more energy delivery algorithms 152, a processor 154, a data storage / retrieval unit 156 (e.g., memory and / or database), and an energy storage subsystem 158 for generating and storing the energy to be delivered. Additional accessories and equipment may be used. The energy delivery device 102 delivers the energy provided by the waveform generator 104 according to one or more energy delivery algorithms 152.

[0381]

[0423] In this embodiment, the energy delivery device 102 comprises a rigid shaft 106, an electrode body 108, and a handle 105. Typically, the electrode body 108 comprises one or more electrodes. The shaft 106 is rigid enough to pass through the surface of the eye so that its distal end enters the internal portion of the eye. In this embodiment, the shaft 106 has a lumen through which the electrode body 108 passes, such that at least a portion of the electrode body 108 extends beyond the distal end of the shaft 106. In this embodiment, the electrode body 108 is in the form of a conductive rod or wire acting as an electrode, sized to pass through the lumen in the shaft 106, and configured to pass through the lumen in the shaft 106. In this embodiment, the handle 105 comprises an actuator 132 that can be operated to operate the electrode body 108. For example, in this embodiment, the actuator 132 comprises a button that moves the electrode body 108 forward and backward from the distal end of the shaft 106. This allows the electrode body 108 to retract while the shaft 106 is inserted into the eye, and to advance the electrode body 108 toward or into the target tissue region once the shaft 106 is in the desired position. It will be understood that other types of actuators 132, including slides, ratchets, knobs, dials, sensors, etc., may also be used. Once the electrode body 108 is exposed in the desired position, pulsed electric field energy is delivered from the generator 104 through the electrode body 108 to the target tissue region. In this embodiment, the shaft 106 is non-conductive or insulated so that the energy provided by the electrode body 108 originates from the exposed portion of the electrode body 108.

[0382]

[0424] It will be understood that many of the energy delivery devices 102 described throughout this specification are configured to provide unipolar energy delivery. In such cases, the return electrode is positioned on the patient's surface, for example, on the torso, buttocks, or lower limbs. When treating the eyes, the return electrode may be positioned closer to the eye, such as on the surface of the eye. It will also be understood that in some embodiments, the energy delivery device 102 is configured to deliver energy in a bipolar or multipolar configuration. In such embodiments, the energy delivery device 102 comprises two or more electrode bodies 108 that work together in a bipolar or multipolar configuration, or the electrode bodies are configured to function in a bipolar or multipolar manner, for example, two or more electrodes or a portion of the electrode bodies 108 acting as a bipolar pair or a multipolar set. Similarly, in some embodiments, a portion of the bipolar pair or multipolar set is located on a separate device. As a result, a separate device that delivers or receives current will be described as an energy delivery device 102, even if it is used to receive the current. Similarly, the energy delivery body 108 and the electrode body 108 are interchangeable. This is for the sake of naming simplification, as the delivery or reception function may change with changes in the polarity of the bipolar pair or multipolar set. It will be understood that the energy delivery body and electrode body may comprise one or more electrodes.

[0383]

[0425] In some embodiments, it will be understood that the energy delivery device 102 also delivers molecules that will be taken up by target cells. However, in other embodiments, the molecules are delivered by a separate device, such as a hypodermic needle. If necessary, the molecules may be delivered by both the energy delivery device and the separate device.

[0384]

[0426] Energy, such as pulsed electric field (PEF) energy, is provided by a generator 104 and delivered to intraocular tissue via at least one electrode body 108. These electrical pulses are provided by at least one energy delivery algorithm 152. In some embodiments, each energy delivery algorithm 152 defines a signal having a waveform comprising a series of pulses. The algorithm 152 specifies the parameters of the signal, for example, energy amplitude (e.g., voltage), as well as the duration of the applied energy, which consists of the number of pulses, pulse width, and delay between pulses. In some embodiments, one or more electrodes are small and tend to dissipate a large amount of energy around them. Therefore, it is desirable to deliver energy optimally. In some embodiments, a large DC link capacitance by a transistor half-bridge is utilized to provide efficient delivery pulses in such cases. This is sometimes preferred with respect to pulse voltages delivered by a power amplifier (limited bandwidth) or an exponential attenuation generator. In some embodiments, a feedback loop based on sensor information and auto-shutoff specifications may be included.

[0385]

[0427] In some embodiments, biphasic pulses may be used. In such embodiments, additional parameters may include the switching time between polarities in the biphasic pulse and the dead time between biphasic cycles. In some cases, biphasic waveforms are advantageous for reducing muscle stimulation in the patient. This is particularly important in applications where even slight movement of the electrode body can easily negate the therapeutic effect. Biphasic waveforms require rapid changes in the phase / polarity of the signal to minimize nerve activation during transitions between polarities. Multiple high-speed switching elements (e.g., MOSFETs, IGBT transistors) are desirable and may be employed and configured, for example, in an H-bridge or full-bridge structure.

[0386] C. Subretinal bleb

[0428] As previously mentioned, molecules can be delivered to the eye by various mechanisms, including injection into the vitreous cavity (or the region between the lens and the retina), injection into the suprachoroidal space (the potential space between the sclera and choroid that traverses the periphery of the posterior segment of the eye), and / or injection into a subretinal bleb (a small region formed by separating the retinal pigment epithelium (RPE) that supports the lamina retina (LRC)). Figure 30 illustrates an embodiment of the procedure for forming a subretinal bleb RB, which can be described as follows.

[0387]

[0429] Before the surgery, the pupil of eye E is dilated and topical antibiotic eye drops are applied. The surgery is usually performed under general anesthesia with the addition of a local anesthetic.

[0388]

[0430] In this embodiment, the subretinal bleb is described as being formed using a subretinal injection device 201. Such a device 201 is configured to deliver a solution, such as a molecular solution, in order to form the bleb. In such an embodiment, energy is provided by an energy delivery device 102, which is a separate device. However, it will be understood that this procedure may be carried out using an energy delivery device 102 that has the capability to deliver a solution, such as a molecular solution. In such a case, the bleb is formed using the energy delivery device 102, and energy is delivered using the same device.

[0389]

[0431] In this embodiment, the subretinal injection device 201 is prepared on a sterile surgical field. The injection device 201 and any elongated tubular material are immobilized in a syringe (not shown) containing molecules (e.g., plasmids, RNA, oligonucleotides, deoxyoligonucleotides, antisense oligonucleotides, or proteins). The volume of molecules available for injection is determined.

[0390]

[0432] A lid speculum is inserted, and a standard three-port transsquamative vitrectomy is performed. The tip of the infusion cannula is directly visualized through the pupil to confirm its position within the vitreous cavity. At this point, infusion should be continued until the surgery is complete.

[0391]

[0433] Core vitrectomy is performed at a high cutting speed and low aspiration setting. The vitreous humor is removed as completely as possible. After core vitrectomy is complete, complete posterior vitreous detachment (PVD) is confirmed. At this point, the vitreous cortex is no longer attached to the macular region. The remaining mobile vitreous humor is then removed as completely as possible with vitrectomy instruments.

[0392]

[0434] The retina is examined before subretinal injection of the molecule. If retinal damage is found, it is treated. The molecule injection is performed in two stages. First, the retina is indented and the tip of the device is positioned so that the tip is covered by the retina. This position should be far enough away from the fovea to minimize mechanical stress on the fovea caused by the injection. A small amount of molecule is injected to confirm that the tip is not obstructed and is properly positioned. Next, if a bleb is raised, the molecule is injected to deliver a total volume of up to 0.3 mL or any intended volume (e.g., up to 1 mL). If no bleb forms during the test injection, or if the bleb does not increase in size with further injection, the cannula tip is repositioned away from the original retinal incision site, and this series of operations is repeated. The second retinal incision should be far enough away that any injected additional material does not connect to the first retinal incision site and flow back. After the entire contents of the syringe have been dispensed, the infusion device 201 is held in place (to allow the remaining contents of the syringe to be delivered). After this, the infusion device 201 is withdrawn.

[0393]

[0435] The subretinal bleb RB provides space for positioning molecules adjacent to photoreceptors in retinal pigment epithelial cells (RPEs) and the lamina retinata (LRC), and / or for positioning an electrode 108 for delivering PEF energy. In some embodiments, this proximity increases the cells' ability to take up molecules upon delivery of pulsed electric field energy. In some embodiments, the nature of the pulsed electric field energy determines which cells take up the molecules and the amount of uptake. Uptake can be controlled or influenced by various factors, such as the selection of signal parameters, the waveform of the signal, the polarity of the electrodes, the position of the electrodes, the type of molecule, and the impedance characteristics of the anatomical environment. Thus, in some embodiments, the photoreceptors in the LRC take up the molecules; in other embodiments, the RPEs take up the molecules; and in yet another embodiment, both the photoreceptors and the RPEs take up the molecules. Similarly, in other embodiments, other retinal layers, ocular tissues, or peripheral regions may be targeted. In some embodiments, uptake is selective based on signal parameters, and it will be understood that, for example, one set of parameters may induce uptake by one part of the anatomy (e.g., LRC), another set of parameters may induce uptake by another part of the anatomy (e.g., RPE), and yet another set of parameters may induce uptake by two or more parts of the anatomy (e.g., LRC and RPE). Similarly, the cells that take up molecules can also be changed by reversing or changing the polarity of the electrode bodies.

[0394]

[0436] Figure 31 illustrates the positioning of an embodiment of the energy delivery device 102 into the eye E such that the electrode body 108 is positioned within the subretinal bleb RB. In this embodiment, the shaft 106 passes through the surface of the eye E and the retina R, and the handle 105 is located outside the eye E. Although the energy delivery device 102 is shown to enter the eye E at a different location than the injector 201, it will be understood that the energy delivery device 102 may be introduced into the eye E through the same opening when the injector 201 is removed. Once the energy delivery device 102 is desiredly positioned, pulsed electric field energy is delivered from the generator 104 via the electrode body 108. In this example, the electrode body 108 has a positive charge, and the return electrode outside the eye (e.g., a remote return electrode) is positioned on the patient's surface and has a negative charge. In this embodiment, the electrode 108 delivers energy to the subretinal bleb RB in a unipolar form, and this energy flows out of the subretinal bleb RB (indicated by the arrow) to negatively drive molecules 110 within the bleb RB to the retina R and, optionally, other structures of the eye E. It will be understood that the direction in which the energy drives molecules 110 depends, to give a few examples, on the charge of molecules 110, the polarity of the electrodes, and the position of the electrodes. Other combinations and results will be described in later sections. The energy delivery device 102 is then removed.

[0395]

[0437] Figures 32A and 32B illustrate the molecules 110 that entered the RPE upon application of pulsed electric field energy. In particular, Figure 32A illustrates eye E in which a subretinal bleb RB containing the molecules was formed (indicated by an asterisk *). Energy is applied so that retinal cells can take up the molecules. Figure 32B illustrates the molecules 110 present in the RPE.

[0396]

[0438] Before closing the scleral incision for intravenous fluid administration, clamp the IV line to prevent fluid from entering the choroid. Suture the incision closed. Next, administer a subconjunctival injection of 0.5 mL of 4 mg / mL dexamethasone solution (for acute inflammation) and 0.5 mL of antibiotic solution. Apply ointment to the eye surface and cover with an eye patch, and protect the eye where the subretinal injection was administered with an eye shield. D. Examples of delivery options

[0397]

[0439] To give a few examples, it will be understood that various delivery options can be used, such as molecular delivery at various positions, electrode placement at various positions, different types of electrode arrangements, different types of waveforms, and various combinations of signal parameters. At least some combinations are described herein, but are not limited thereto.

[0398]

[0440] Table 8 shows various combinations of delivery options. Here, molecule 110 is delivered to various locations, e.g., subretinal bleb, vitreous cavity, or a combination of subretinal bleb and vitreous cavity. Similarly, electrode 108 can be positioned in various locations, e.g., subretinal bleb, vitreous cavity, adjacent to the cornea, or in the periocular space (e.g., subconjunctival, sub-Tenon, or posterior). In some cases, it will be understood that the subretinal bleb may be replaced with the suprachoroidal space in these combinations when so desired. Similarly, the suprachoroidal space may be used in combination with one or more blebs for molecule and / or energy delivery. Injections of 10–50 μL into the suprachoroidal space have been demonstrated to be well-tolerated with a low risk of ocular complications, and in some cases, injections up to 1 mL are possible. Suprachoroidal delivery may be particularly useful when targeting the retinal pigment epithelium.

[0399]

[0441] The suprachoroidal space has long been known as a latent space between the choroid and the sclera. The inner border of the choroid, which is Bruch's membrane, is dense, while the outer border is a transitional zone consisting of several fibrous lamellae of varying thickness. The suprachoroidal space has been shown to be present in approximately 50% of people over the age of 50. The presence of the suprachoroidal space correlates with hyperopic refractive errors and is generally absent in the eyes of young, healthy individuals. In hyperopia, it is theorized that compression of vortex veins by the sclera increases hydrostatic pressure, leading to occult suprachoroidal exudation, which becomes visible as a small amount of fluid in the suprachoroidal space during imaging. It has also been proposed that the detection rate is higher in the elderly because, with age, protein leakage from choroidal vessels into the suprachoroidal space increases, and osmotic pressure also rises. The suprachoroidal space is also present in various patients with specific conditions. Therefore, in some cases, the suprachoroidal space may be utilized as a delivery site.

[0400]

[0442] [Table 8]

[0401]

[0443] These various combinations are described and illustrated in more detail herein. It will be understood that numerous combinations exist, and only a portion of them are provided herein. Similarly, two or more subretinal blebs may form in one eye. Thus, molecules and / or energy delivery devices may be delivered to two or more blebs for further combinations. Even the combinations listed in Table 1 include subcombinations, such as having two variations in the charge (positive and negative) of each bipolar pair. Similarly, the location of molecule 110 and / or the location of electrode body 108 within its anatomical location may also affect the outcome. For example, positioning a molecule in a certain region of the vitreous cavity may yield different results than a molecule positioned in a different region of the vitreous cavity. Or, molecule 110 concentrated in a certain region of the vitreous cavity may yield different results than a molecule diffused throughout the entire vitreous cavity. Similarly, anatomical features may affect outcomes such as the innate impedance of certain tissues that may influence the movement of molecule 110. Furthermore, various types of waveforms can cause the molecule 110 to move in different ways (including moving at different velocities and trajectories). For example, some waveforms induce translation, while others induce rotation, oscillation, or a combination of these and translation. It will be understood that the examples provided herein are partial and not limiting.

[0402]

[0444] Figure 33 illustrates an embodiment in which molecule 110 is delivered to a subretinal bleb RB, and an electrode 108 is positioned within the subretinal bleb RB for unipolar energy delivery thereto. Here, the molecule is negatively charged, the electrode 108 is negatively charged, and the remote return electrode is positively charged. This typically directs the molecule away from the electrode 108 to, for example, the retinal pigment epithelium (RPE) and the layer of rods and cones (LRC). In some cases, guidance to the RPE or LRC can be achieved by the placement of the electrode 108 within the subretinal bleb RB, the selection of the waveform, or other factors. In a similar embodiment, molecule 110 is also added to the vitreous cavity V. This region between the lens and the retina will be referred to herein as the vitreous cavity V, regardless of whether this region is filled with vitreous fluid or another solution. The molecule 110 within the vitreous cavity 110 is typically delivered to a portion of the retina further away from the subretinal bleb RB. It will be understood that molecule 110 may be specifically located in the vitreous cavity V to facilitate its migration to a specific region of the retina.

[0403]

[0445] Figure 34 illustrates an embodiment in which molecule 110 is delivered to a subretinal bleb RB, and an electrode 108 is positioned in the vitreous cavity V of the eye E for unipolar delivery thereto. Here again, the molecule is negatively charged, the electrode 108 is negatively charged, and the remote return electrode is positively charged. This typically delivers molecule 110 away from the electrode 108, for example, to the retinal pigment epithelium (RPE). Depending on the orientation and polarity of the electrodes, it will be understood that molecule 110 can be pushed toward and into the retinal pigment epithelium (RPE) cells. In some embodiments, by changing the polarity of the pulses with each pulse or after multiple pulses (biphasic) (e.g., 4 up, 4 down...2 up, 2 down, etc.), molecule 110 is driven in one direction and then in another, thereby enabling targeting of both retinal pigment epithelium (RPE) cells and the lamina ligament (LRC). In similar embodiments, molecule 110 is also added to the vitreous cavity V. In such embodiments, the molecules 110 in the vitreous cavity 110 are typically delivered to the LRC. It will be understood that the molecules 110 may be specifically positioned in the vitreous cavity V to facilitate their migration to a specific region of the retina. In some embodiments, it will be understood that various types of molecules 110 are placed in the bleb and the vitreous cavity.

[0404]

[0446] Figure 35 illustrates an embodiment in which molecule 110 is delivered to the vitreous cavity V, and an electrode 108 is positioned in the vitreous cavity V for unipolar delivery thereto. In such a case, since the desired access is direct access to the vitreous cavity V, no subretinal bleb RB is formed. Here again, molecule 110 is negatively charged, electrode 108 is negatively charged, and the remote return electrode is positively charged. This typically delivers the molecule away from electrode 108, for example, into the LRC. In some cases, it will be understood that molecule 110 is concentrated in a specific region or region of the vitreous cavity V prior to energy delivery. For example, in some embodiments, molecule 110 is delivered to a specific location in the vitreous cavity V, for example, close to a target tissue region, and / or to a specific concentration to provide enhanced and / or more predictable delivery of molecule 110 to target cells. In such cases, it may be desirable for the electrode 108 to be placed in the vitreous cavity V in relation to the concentration of the molecule 110 in order to optimize delivery. In some embodiments, this is in the same location in the vitreous cavity V, and in other embodiments, it is adjacent to such a location in order to guide the molecule 110 in a particular direction. For example, in some embodiments, the molecule 110 is positioned adjacent to the LRC, and the electrode 108 is positioned adjacent to the molecule 110 so that the energy of the current drives the molecule 110 away from the electrode 108 and into the LRC.

[0405]

[0447] In other embodiments, energy is delivered in a bipolar manner, for example, using two separate energy delivery devices 102, or using a single energy delivery device 102 having one or more pairs of electrodes. For example, Figure 36 illustrates a molecule 110 located in a subretinal bleb RB, and two electrodes located in the eye E that deliver energy from one to the other in a bipolar manner. Referring to Figure 36, the molecule 110 is shown in the subretinal bleb RB, and a first energy delivery device 102' having a first electrode 108' is shown, inserted into the eye E so that the first electrode 108' is located in the subretinal bleb RB. In this embodiment, a second energy delivery device 102" having a second electrode 108" is shown, inserted into the eye E so that the second electrode 108" is located in the vitreous cavity V of the eye E. In this embodiment, current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108" When both bipolar electrodes are used in this configuration in the eye, negatively charged molecules (e.g., DNA) can be pushed towards and into, for example, the vitreous layer LRC. It will be understood that the position of the second electrode 108" in the vitreous cavity V can be selected to elicit a specific effect (e.g., to deliver molecules 110 to a specific part of the LRC, or to increase the scale of its drive). In a similar embodiment, the second electrode 108" is positioned on the cornea C rather than in the vitreous cavity V. The results may be similar to those described in relation to Figure 36. However, the energy field may cause a slight increase in delivery to the RPE, while the majority of molecules 110 will continue to be delivered to the LRC.

[0406]

[0448] Figure 37 illustrates an embodiment similar to that in Figure 36, utilizing two electrodes (one in the subretinal bleb RB and the other in the vitreous cavity V). However, here the molecule 110 is located in the vitreous cavity V, and the polarity of the electrodes is reversed. Thus, a second energy delivery device 102" having a second electrode 108" is shown, inserted into the eye E such that the first electrode 108' is located in the vitreous cavity V and the second electrode 108" is located in the subretinal bleb RB. Current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108". Here again, the molecule is negatively charged, the first electrode 108' is negatively charged, and the second electrode 108'' is positively charged. This typically causes the molecule to be delivered away from the first electrode 108' (e.g., into the LRC). Again, it will be understood that the positions of the electrodes 108', 108'' and the molecule 110 within the vitreous cavity V can be selected to elicit a specific effect (e.g., to deliver the molecule 110 to a specific part of the LRC, or to increase the scale of its drive). In a similar embodiment, the first electrode 108' is located on the cornea C. The results may be similar to those described in relation to Figure 37.

[0407]

[0449] Figure 38 illustrates an embodiment similar to that of Figure 37, but the molecule 110 is located in both the subretinal bleb RB and the vitreous cavity V. In this case as well, the first electrode 108' is located in the vitreous cavity V, and the second electrode 108'' is located in the subretinal bleb RB. Current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108''. In some embodiments, the molecule is negatively charged, the first electrode 108' is negatively charged, and the second electrode 108'' is positively charged. This typically drives the molecule 110 away from the first electrode 108. Thus, the molecule 110 in the vitreous cavity V is driven to the LRC, and the molecule in the subretinal bleb RB is driven away from the electrode 108' to, for example, the LRC or RPE. In some embodiments, it will be understood that various types of molecules 110 are placed in the bleb and the vitreous cavity.

[0408]

[0450] Figure 39 also illustrates bipolar energy delivery to the eye. Here, a first energy delivery device 102' having a first electrode 108' is shown, inserted into the eye E such that the first electrode 108' is positioned within the vitreous cavity V. In this embodiment, a second energy delivery device 102” having a second electrode 108” is shown, inserted into the posterior ossicular septum RBS of the eye E. The posterior ossicular septum RBS is located on the back side of the eye E, near the optic nerve ON within the muscular cone MC. Current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108”. In this embodiment, the molecule 110 is positioned within the subretinal bleb RB. When devices 102', 102” are used in this configuration, negatively charged molecules (e.g., DNA) are typically pushed toward and into the RPE. If the polarities of electrodes 108' and 108'' are reversed, it will be understood that negatively charged molecules can be pushed toward and into the LRC. In a similar embodiment, the first electrode 108' is located on the cornea C, and the second electrode 108'' is located in the posterior ocular space RBS. The results may be similar to those described in relation to Figure 39, but in some cases, the energy field may be more homogenized. This may result in a more uniform transfer of molecules 110 to the RPE.

[0409]

[0451] Referring to Figure 40, molecule 110 is again shown within the subretinal bleb RB, but here a first energy delivery device 102' having a first electrode 108' is shown, inserted into eye E so that the first electrode 108' is positioned within the subretinal bleb RB. In this embodiment, a second energy delivery device 102” having a second electrode 108” is shown, inserted into the posterior ocular space RBS of eye E. Current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108”. When devices 102', 102” are used in this configuration, negatively charged molecules (e.g., DNA) can be pushed toward and into the RPE, for example, but a small amount can be pushed toward and into the LRC.

[0410]

[0452] Figure 41 illustrates a similar embodiment, but here the molecule 110 is located in the vitreous cavity V rather than the subretinal bleb RB. In this embodiment, energy is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108''. When devices 102', 102'' are used in this configuration, negatively charged molecules (e.g., DNA) are repelled by the first electrode 108' but follow the electric field around the first electrode 108' toward the second electrode 108''. Thus, the molecule 110 can be delivered toward the sides of the retina rather than directly to the portion of the retina covering the surface of the subretinal bleb RB. This can be beneficial when treatment of specific portions of the retina in these regions is desired.

[0411]

[0453] Figure 42 illustrates an embodiment similar to those in Figures 40 and 41, but here the molecule 110 is located in both the subretinal bleb RB and the vitreous cavity V. Current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108''. In some embodiments, the molecule is negatively charged, the first electrode 108' is negatively charged, and the second electrode 108'' is positively charged. This typically drives the molecule 110 away from the first electrode 108. Thus, the molecule 110 in the vitreous cavity V is delivered to the LRC, and the molecule in the subretinal bleb RB is delivered to the RPE. In some embodiments, it will be understood that various types of molecules 110 are placed in the bleb and the vitreous cavity.

[0412]

[0454] Figure 43 illustrates an embodiment in which molecule 110 is delivered to the vitreous cavity V and the first electrode body 108' is positioned within the vitreous cavity V. In this case, since the desired access is direct access to the vitreous cavity V, no subretinal bleb RB is formed. In this embodiment, the second electrode body 108'' is positioned in contact with the cornea C. It is recognized that various different designs can be used to position the electrode body in contact with the cornea C. Various scleral-type contact lens electrodes may be used. Examples include the Burian-Allen contact lens electrode and the DTL® (Diagnosys LLC) fiber electrode, which are the most frequently used electrodes in the United States for recording electroretinograms (ERGs), and which can be used to deliver the currents described herein. Other examples include gold ring electrodes and jet contact lens electrodes. Other types of devices include tweezerstrodes or microscopes used around the eye.

[0413]

[0455] The current is delivered in a bipolar manner to the first electrode 108' and flows to the second electrode 108''. Here again, the molecules are negatively charged, the first electrode 108' is negatively charged, and the second electrode 108'' is positively charged. Therefore, molecules 110 positioned between the first electrode 108' and the LRC can be driven away from the first electrode 108' toward the LRC and then follow the electric field that bends toward the second electrode 108''. Thus, these molecules 110 are fed toward and into the LRC. It will be understood that this effect can be enhanced by positioning the molecules 110 closer to the LRC and positioning the first electrode 108' between the molecules 110 and the second electrode 108''. In a similar embodiment, it would be understood that the second electrode body 108" is located in the posterior ossicular space (RBS) rather than on the cornea C. In this modification as well, the molecule 110 is delivered to the LRC, which may result in a stronger drive depending on the circumstances.

[0414] E. Examples of devices

[0456] Energy can be delivered by various energy delivery devices 102. Typically, the energy delivery device 102 comprises an elongated shaft having a distal end that can be advanced with the body to target tissue, and an electrode body 108 positioned near the distal end. The electrode body 108 comprises one or more electrodes that deliver PEF energy to the target tissue. Such a device is configured to access target tissue on or within the eye E. In some embodiments, such a device is configured to access target tissue within or near the retina R.

[0415]

[0457] As previously mentioned, in some embodiments, the energy delivery device 102 is configured to deliver a solution, such as a solution containing molecules 110, to target tissue. Figures 44A to 44B illustrate such embodiments. Figure 44A illustrates an embodiment of the energy delivery device 102, comprising a rigid shaft 106, an electrode body 108, and a handle 105. The shaft 106 is rigid enough to pass through the surface of the eye so that its distal end enters the internal portion of the eye. In this embodiment, the shaft 106 has a lumen through which the electrode body 108 passes, such that at least a portion of the electrode body 108 extends beyond the distal end of the shaft 106. In this embodiment, the energy delivery device 102 includes an injection port 300 that is fluidly connected to the lumen of the shaft 106. In some embodiments, the injection port 300 includes a Luer fitting 302 for attaching a syringe. Typically, a syringe containing the desired solution is attached to the Luer fitting 302. When solution delivery is desired, the electrode body 108 is retracted to allow passage through the lumen of the shaft 106. In this embodiment, the handle 105 includes an actuator 132 that can be operated to manipulate the electrode body 108. In this embodiment, the actuator 132 includes a slide that advances and retracts the electrode body 108 from the distal end of the shaft 106. This allows the electrode body 108 to be retracted.

[0416]

[0458] Subsequently, the solution is injected and discharged into the target tissue region through the distal end of the shaft 106. The electrode body 108 is then advanced through the lumen by sliding the actuator 132. In some embodiments, it will be understood that the lumen is sized such that the solution is injected through it while the electrode body 108 is in place. This makes it possible to deliver both the solution and energy to the target tissue region simultaneously.

[0417]

[0459] Figure 44B provides an additional diagram of an embodiment of an energy delivery device 102 having an injection port 300 that is fluidly connected to the lumen of a shaft 106. In this diagram, a syringe 304 that can be attached to a Luer fitting 302 is shown. Similarly, a cable 310 extending from a handle 105 is shown, where the cable 310 has a fitting 312 configured to connect to a generator 104. This delivers electrical energy to the energy delivery device 102, in particular to the electrode body 108.

[0418]

[0460] It will be understood that the energy delivery device 102, particularly the electrode body 108, can take on various forms. Figure 45 illustrates an embodiment of the energy delivery device 102 showing a close-up of its distal end. Here, the energy delivery device 102 comprises a shaft 106 having a lumen 107 through which the electrode body 108 extends. In this embodiment, the electrode body 108 comprises a conductive rod or wire 320. In this embodiment, the wire 320 has a pointed distal end configured to penetrate tissue, but it will be understood that the distal end can have various shapes, including a blunt or rounded shape.

[0419]

[0461] In this document, the terms “a” or “an” are used to include one or more, independently of any other instance or usage of “at least one” or “one or more,” as is common in patent documents. In this document, the term “or” is non-exclusive unless otherwise indicated, or “A or B” is used to mean “including A but not B,” “including B but not A,” and “including A and B.” In this document, the terms “including” and “in which” are used as plain English equivalents of the terms “comprising” and “wherein.” Furthermore, in the following claims, the terms “including” and “comprising” are open-ended, meaning that a system, device, article, composition, formulation or process containing elements other than those listed before this term in the claim is still considered to be within the scope of that claim. Furthermore, in the following claims, terms such as "first," "second," and "third" are used merely as labels and are not intended to impose numerical requirements on the subject.

[0420]

[0462] The above description is intended to illustrate, not to limit. For example, the examples (or one or more embodiments thereof) described above may be used in combination with each other. Those skilled in the art may use other embodiments when reviewing the above description. The abstract is provided in accordance with 37 1.72(b) of the U.S. Federal Rules Code so that the reader may quickly grasp the essence of the technical disclosure. The abstract is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Furthermore, in the above detailed description, various features may be grouped together to streamline the disclosure. This should not be interpreted as meaning that disclosed unclaimed features are essential to any claim. Rather, the subject matter of the invention may exist in fewer features than all the features of a particular disclosed embodiment. Accordingly, the following claims are incorporated into the detailed description as examples or embodiments, and each claim stands on its own as a distinct embodiment, and such embodiments are intended to be combined with each other in various combinations or permutations. The scope of the invention should be determined by reference to the appended claims, along with the entire scope of equivalents to which such claims are granted.

Claims

1. A system for transferring molecules to target tissue cells within the patient's body, An energy delivery device having at least one energy delivery body configured to be positioned near the target tissue cells within the body, A generator electrically connected to the at least one energy delivery body, wherein the generator includes at least one energy delivery algorithm configured to provide a deliverable electrical signal to the at least one energy delivery body in order to deliver pulsed electric field energy that causes at least one of the molecules to enter at least one of the target tissue cells, and A system comprising, wherein the electrical signal comprises a series of pulses, the series of pulses together having a balance of charge between positive amplitude on time and negative amplitude on time, the series of pulses having at least one pulse having a fundamental pulse width of sufficient length to cause muscle stimulation in the body, the at least one pulse comprising a plurality of interval pulses, the plurality of interval pulses having interval delays between them sufficient to at least reduce the muscle stimulation, and the at least one interval pulse having an on time not exceeding 50 μs.

2. The system according to claim 1, wherein the series of pulses together have a balance of positive amplitude on-time and negative amplitude on-time that is sufficient to avoid ablation of the target tissue cells and / or muscle stimulation within the body.

3. The system according to claim 1, wherein at least one of the series of pulses has a voltage in the range of 10 to 500 V.

4. The system according to claim 1, wherein at least one of the series of pulses has a pulse duration in the range of 0.5 to 200 ms.

5. The system according to claim 1, wherein each interval delay has a duration in the range of 1 to 100,000 μs.

6. The system according to claim 1, wherein each interval pulse has an on-time not exceeding 10 μs.

7. The system according to claim 1, wherein the series of pulses is a series of biphasic pulses, at least one biphasic pulse having a cycle length of 0.01 to 10 μs, the at least one biphasic pulse comprising a plurality of interval pulses, the plurality of interval pulses having interval delays between them.

8. The system according to claim 7, wherein the series of biphasic pulses are grouped into packets having inter-packet delays.

9. The system according to claim 1, wherein the molecule comprises plasmids, DNA, synthetic DNA vectors, RNA, nucleic acid-based molecules, antisense oligonucleotides, oligomeric molecules, ribozymes, ribonucleoproteins, CRISPR, recombinant proteins, proteolytically targeted chimeras, zinc finger nucleases or transcription activator-like effector nucleases, proteins and / or materials that induce genetic or epigenetic changes in cellular behavior.

10. The system according to claim 1, wherein the molecule comprises at least one gene greater than 10 kb.

11. The system according to claim 1, wherein the target tissue cells include retinal cells.

12. The system according to claim 1, wherein the energy delivery device comprises a shaft, the shaft having one or more tines extendable therefrom.

13. The system according to claim 12, wherein the one or more tines at least partially define the at least one energy deliverer and at least one additional energy deliverer, and the generator includes an energy delivery algorithm configured to provide different electrical signals to the at least one additional energy deliverer.

14. A system for transferring molecules to target tissue cells within a patient's body, An energy delivery device having at least one energy delivery body configured to be positioned near the target tissue cells within the body, A generator electrically connected to the at least one energy delivery body, wherein the generator includes at least one energy delivery algorithm configured to provide a deliverable electrical signal to the at least one energy delivery body in order to deliver pulsed electric field energy that causes at least one of the molecules to enter at least one of the target tissue cells, and The electrical signal comprises a series of pulses, and the series of pulses together have a balance of charge due to positive amplitude on time and negative amplitude on time. The energy delivery device comprises a shaft, the shaft having one or more tines extendable therefrom, the one or more tines at least partially defining the at least one energy delivery body and at least one additional energy delivery body, and the generator includes an energy delivery algorithm configured to provide different electrical signals to the at least one additional energy delivery body, the different electrical signals generating energy that causes ablation of at least one target tissue cell, the system.

15. The system according to claim 14, wherein one or more tines are configured to provide ablation of a first region of the target tissue cell and to provide transfer of molecules to a second region of the target tissue cell, the second region at least partially surrounding the first region.

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