Direct Intraparenchymal Injection Research Articles

In Vivo Gene Therapy for Mucopolysaccharidosis Type III (Sanfilippo Syndrome): A New Treatment Horizon.

For most lysosomal storage diseases (LSDs), there is no cure. Gene therapy is an attractive tool for treatment of LSDs caused by deficiencies in secretable lysosomal enzymes, in which neither full restoration of normal enzymatic activity nor transduction of all cells of the affected organ is necessary. However, some LSDs, such as mucopolysaccharidosis type III (MPSIII) diseases or Sanfilippo syndrome, represent a difficult challenge because patients suffer severe neurodegeneration with mild somatic alterations. The disease's main target is the central nervous system (CNS) and enzymes do not efficiently cross the blood-brain barrier (BBB) even if present at very high concentration in circulation. No specific treatment has been approved for MPSIII. In this study, we discuss the adeno-associated virus (AAV) vector-mediated gene transfer strategies currently being developed for MPSIII disease. These strategies rely on local delivery of AAV vectors to the CNS either through direct intraparenchymal injection at several sites or through delivery to the cerebrospinal fluid (CSF), which bathes the whole CNS, or exploit the properties of certain AAV serotypes capable of crossing the BBB upon systemic administration. Although studies in small and large animal models of MPSIII diseases have provided evidence supporting the efficacy and safety of all these strategies, there are considerable differences between the different routes of administration in terms of procedure-associated risks, vector dose requirements, sensitivity to the effect of circulating neutralizing antibodies that block AAV transduction, and potential toxicity. Ongoing clinical studies should shed light on which gene transfer strategy leads to highest clinical benefits while minimizing risks. The development of all these strategies opens a new horizon for treatment of not only MPSIII and other LSDs but also of a wide range of neurological diseases.

Efficient Gene Transfer to the Central Nervous System by Single-Stranded Anc80L65

Adeno-associated viral vectors (AAVs) have demonstrated potential in applications for neurologic disorders, and the discovery that some AAVs can cross the blood-brain barrier (BBB) after intravenous injection has further expanded these opportunities for non-invasive brain delivery. Anc80L65, a novel AAV capsid designed from in silico reconstruction of the viral evolutionary lineage, has previously demonstrated robust transduction capabilities after local delivery in various tissues such as liver, retina, or cochlea, compared with conventional AAVs. Here, we compared the transduction efficacy of Anc80L65 with conventional AAV9 in the CNS after intravenous, intracerebroventricular (i.c.v.), or intraparenchymal injections. Anc80L65 was more potent at targeting the brain and spinal cord after intravenous injection than AAV9, and mostly transduced astrocytes and a wide range of neuronal subpopulations. Although the efficacy of Anc80L65 and AAV9 is similar after direct intraparenchymal injection in the striatum, Anc80L65’s diffusion throughout the CNS was more extensive than AAV9 after i.c.v. infusion, leading to widespread EGFP expression in the cerebellum. These findings demonstrate that Anc80L65 is a highly efficient gene transfer vector for the murine CNS. Systemic injection of Anc80L65 leads to notable expression in the CNS that does not rely on a self-complementary genome. These data warrant further testing in larger animal models.

Magnetic Resonance Imaging-Guided Transplantation of Neural Stem Cells into the Porcine Spinal Cord

Background: Cell-based therapies are a promising treatment option for traumatic, tumorigenic and degenerative diseases of the spinal cord. Transplantation into the spinal cord is achieved with intravascular, intrathecal, or direct intraparenchymal injection. The current standard for direct injection is limited by surgical invasiveness, difficulty in reinjection, and the inability to directly target anatomical or pathological landmarks. The objective of this study was to present the proof of principle for minimally invasive, percutaneous transplantation of stem cells into the spinal cord parenchyma of live minipigs under MR guidance. Methods: An MR-compatible spine injection platform was developed to work with the ClearPoint SmartFrame system (MRI Interventions Inc.). The system was attached to the spine of 2 live minipigs, a percutaneous injection cannula was advanced into the spinal cord under MR guidance, and cells were delivered to the cord. Results: A graft of 2.5 × 10⁶ human (n = 1) or porcine (n = 1) neural stem cells labeled with ferumoxytol nanoparticles was transplanted into the ventral horn of the spinal cord with MR guidance in 2 animals. Graft delivery was visualized with postprocedure MRI, and characteristic iron precipitates were identified in the spinal cord by Prussian blue histochemistry. Grafted stem cells were observed in the spinal cord of the pig injected with porcine neural stem cells. No postoperative morbidity was observed in either animal. Conclusion: This report supports the proof of principle for transplantation and visualization of pharmacological or biological agents into the spinal cord of a large animal under the guidance of MRI.

323. Adenoviral Compartmentalized Liver Transduction Improves Safety and Yields Longerm Transgene Expression

Still unresolved complications associated with adenoviral mediated hepatic-based gene therapy are a strong immunological response with damage to the organ and short-term gene expression. Our group hypothesized that the two components at the core of these drawbacks are: 1) the astronomically high quantities of vector infused and 2) the viremia associated with the conventional routes of vector administration. To stringently manipulate these two variables and assess their effect on adenoviral mediated liver transduction our group implemented compartmentalized liver transduction. Compartmentalized liver transduction is based on known adenoviral spatio-temporal kinetics and is achieved by the direct intra-parenchymal injection of adenovirus into a blood-flow isolated portion of the liver; blood flow is reestablished after viral endocytosis has been completed. Implementing compartmentalized liver transduction in a rat model, gene expression and transgene presence was confined to the site of injection, no inflammation nor liver damage was observed, reporter protein expression with low quantities of vector (10^2) as well as linear dose response curves were documented and prolonged transgene expression was achieved. Our results conclude that compartmentalized liver transduction is a safe and effective mode of vector delivery with a promising future in the field of gene therapy.

Site‐targeted non‐viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ.

Current Advances in Intravascular Administration of Stem Cells for Neurological Diseases: A New Dose of Rejuvenation Injected

Selection of the appropriate administration route is one of the critical steps in a successful therapeutic process. From its beginning, stem cell-based rejuvenation of the central nervous system has been based on intraparenchymal cell transplantation. However, a high invasiveness of the procedure and a focal cell delivery can not be justified for any pathological conditions. This review analyses, compares, and comments on observed differences between the intraparenchymal and intravascular administration route for neurological diseases. A special emphasis has been given to the reported obstacles and specific recommendations have been made. More significant success with intravascular delivery of stem cells for neurological diseases can be achieved with smaller cells, use of dilatators of lung vasculature, blocking of endothelial receptors, less aggressive pretreatment of cells, and permeabilization of the blood-brain barrier. If these requirements are met, intravascular administration represents one of the major options for delivery of stem cells to the aging brain, with obvious advantages over direct intraparenchymal injection.

Hemostasis Effects of Direct Intraparenchymal Injection of Platelets and Fresh Frozen Plasma before Cutting Needle Biopsy in an Animal Model

Purpose To evaluate the effectiveness of direct intraparenchymal injection of platelets or fresh frozen plasma (FFP) into the needle tract before cutting needle biopsy to decrease postprocedural blood loss in pigs with normal and abnormal hemostasis. Materials and Methods A total of 12 Yorkshire-cross pigs were anesthetized and maintained on a respirator. The pigs were divided into three groups: three with normal hemostasis, five treated with warfarin anticoagulation, and four treated with aspirin. Four types of biopsies were performed in the exposed livers and kidneys with 14-gauge Tru-Cut needles. The first was a standard (ie, control) biopsy, and the other three were performed with 2 mL normal saline solution, porcine FFP, or platelet-rich porcine plasma injected into the planned needle tract. Biopsy was then performed in the same needle tract. Blood loss was measured with gauze sponges. Results Significantly decreased postbiopsy blood loss was noted after FFP and platelet injection in the livers and kidneys of all groups except the kidney biopsy group after platelet injection in pigs with normal hemostasis. There was no significant difference in blood loss between the control and saline solution control groups in any of the pigs. Conclusions Local injection of platelets or FFP significantly decreases postbiopsy blood loss in pigs with impaired hemostasis.

Intra-ventricular infusion of rAAV1-EGFP resulted in transduction in multiple regions of adult rat brain: A comparative study with rAAV2 and rAAV5 vectors

Most gene transfer studies conducted in the central nervous system (CNS) with recombinant adeno-associated virus (rAAV) vectors have been carried out by direct intra-parenchymal injection. However, this delivery method usually results in transduction of cells in only a limited region and is quite invasive, which may hamper its potential clinical application. Injection of viral vectors into the cerebrospinal fluid (CSF) may provide an alternative strategy for widespread gene delivery to the CNS via the subarachnoid space. In this study we compared the transduction abilities of rAAV types 1, 2, and 5 when infused directly into the right lateral cerebral ventricle of adult rats. Multiple structures in the vicinity of the lateral ventricle were transduced by rAAV1, but not by rAAV2 or rAAV5 vectors. Double immunolabeling showed that the transduced cells included not only neurons, but also glia. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments demonstrated that rAAV1-mediated EGFP mRNA expression was significantly higher than that induced by either rAAV2 or 5. Our data suggest that intra-ventricular infusion of rAAV1 vectors provides a useful method for broad gene delivery to cells in the adult rat CNS.

Adeno-associated virus-mediated gene transfer to the neonatal brain

For many metabolic diseases, early treatment is necessary to prevent irreversible developmental damage. This is particularly true for childhood diseases that affect the central nervous system (CNS). The development of effective techniques for gene transfer to the neonatal brain would provide a new set of therapeutic options for many of these disorders. Vectors based on adeno-associated virus (AAV) have shown promise as agents for neonatal CNS transduction. In preclinical animal models, a single treatment with AAV vectors at birth has been shown to produce persistent CNS expression of transduced genes into adulthood. Transduction of the neonatal brain has been accomplished by a variety of methods, including direct intraparenchymal injection, intraventricular infusion, and intravenous administration. Of these methods, intraparenchymal injection provides the highest levels of localized activity, while intraventricular infusion results in a more widespread distribution of activity when performed in the neonate. Here we describe a method for direct, intraparenchymal injection of AAV into the neonatal brain. This technique provides a method for investigators to evaluate the effects of in vivo expression of exogenous genes on the process of early brain development.

In vivo control of NMDA receptor transcript level in motoneurons by viral transduction of a short antisense gene

Glutamate receptors play critical roles in normal and pathological processes. We developed an antisense gene delivery strategy to modulate the NMDA type of glutamate receptor. Using transient transfection in vitro and viral mediated gene transfer in vitro and in vivo, the effect of expression of an antisense gene fragment (60 bp) of the NR1 subunit was tested. Immunoblot analysis showed an antisense-concentration-dependent reduction in the NR1 subunit upon transient co-transfection of a plasmid expressing a sense NR1 gene and a plasmid expressing the antisense fragment into COS-7 cells. After recombination into an adenoviral vector, this antisense fragment reduced the amount of endogenous NR1 protein in PC12 cells. Finally, direct intraparenchymal injection of the viral vector into rat spinal cord resulted in diminished NR1 in motor neurons. Our results demonstrate the efficacy of this approach, which combines antisense with viral gene delivery to control the expression of specific genes in vivo. This approach may also be useful in reducing excitatory neurotransmission in vivo, with implications for the treatment of spinal disorders such as amyotrophic lateral sclerosis or chronic pain.