Parkinson's Cure PFFNB2 From Johns Hopkins University: Q and A

PFFNB2 Questions and Answers:

Questions posed by PdCure.org and answered by Dr. Xiaobo Mao, the Johns Hopkins head researcher for PFFNB2, a leading candidate for a cure for Parkinson's disease. These questions and answers from October 2024 pertain to the second generation of PFFNB2.

PFFNB2 is a gene therapy which uses a genetically modified AAV virus which has the capability of coding the creation of a nanobody which targets alpha-synuclein proteins for degradation. This modified AVV virus mechanism of treatment allows for its operation within the blood-brain-barrier and within the neuron cell walls. The nanobody does not have to pass through the blood-brain-barrier or through the cell walls as it is generated within as defined by the RNA of the virus,

1. Is using the genetically engineered AAV virus the only way to deploy and engage nanobodies inside a neuron?
    
   Response: Good question, currently, this is the best approach due to its ability to cross the blood-brain barrier and provide long-term expression. While alternative delivery methods could be explored, these are generally less efficient than AAV in terms of achieving intracellular expression, lower delivery efficiency and reduced ability to target neurons specifically. Thus, while not the only option, we believe, AAV is most practical and effective approach for deploying nanobodies intracellularly in neurons.
    
   
2. Are there any steps being taken to avoid immunogenicity?    For example: using multiple AAV serotypes (AVV9, AVVrh.10), anti-adjuvants, immunosuppressive drugs (e.g. corticosteroids), etc.   If not, won't the patient develop an immunity to the AAV virus preventing any redosing or second use of AAV?
    
   Response: Good question, that is true that using AAV will cause immunogenicity and prevents any redesign of the same AAVs. One solution to this is that AAV expression can be long lasting in the brain (like years), so hopefully one dosage would be sufficient. If a second dosage is really needed, we would need to change the serotype of the AAV to something different or co-administer immunosuppressive drugs. Immunogenicity outcomes for AAV gene therapy are varied in ongoing clinical trials, some showing no to mild immunogenicity and some needed immunosuppressants.
    
   
3. Can the PFFNB-2 treatment operate on intracellular alpha-synuclein aggregates if administered as a nanobody without employing the AAV virus?
    
   Response: Recombinant PFFNB2 protein administered via IV injection will mostly target the extracellular alpha-synuclein aggregates, which could also inhibit the pathology progressions, which we have some preliminary data for. However, the extracellular PFFNB2 probably won’t have much effect on the intracellular alpha-synuclein aggregates. Major hurdles in using exogenously purified protein is crossing blood brain barrier, stability once injected as well as PFFNB uptake efficacy by neurons. Without AAV, delivering the PFFNB-2 nanobody intracellularly could be less effective due to challenges in crossing cellular membranes.
    
   
4. In your published paper in Nature about the first generation PFFNB-2, in the "Discussion" section it states: "More study is needed to evaluate the efficacy of exogenously applied PFFNB2 in preventing the neuronal uptake of α-syn PFF and subsequent α-syn propagation. If recombinant PFFNB2 can inhibit the effects of exogenous α-syn PFF when administered extracellularly, we will then modify the AAV-PFFNB2 system to secret PFFNB2 to the extracellular space." Has a study been conducted on the extracellular use of PFFNB-2?
    
   Response: We expressed and purified recombinant PFFNB2 with maltose binding protein (MBP) fusion (MBP-PFFNB2) and added MBP-PFFNB2 to the primary cortical neurons treated with PFF. Our results show that MBP-PFFNB2 can significantly inhibit the pS129 pathology. We also tested treatment of recombinant PFFNB2 to primary cortical neurons in vitro and observed that exogenously applied PFFNB2 prevents the neuronal uptake of biotin labelled α-syn PFF. To target both intracellular and extracellular αS seeds to effectively block αS pathogenesis, Currently, Wang lab is in process of developing and characterizing bifunctional nanobody to express both intracellular PFFNB2-E3 and secreted PFFNB2 with the intent to achieve enhanced efficacy in inhibiting cell-to-cell transmission (i.e., uptake, propagation) of pathogenic αS compared to only intracellular PFFNB2. We call this new reagent dual-function-PFF Binding Proteins (dfPFFBP)
    
   
5. In your published paper in Nature about the first generation PFFNB-2, in the "Discussion" section it states: "Further bioengineering efforts on PFFNB2 will also be necessary to improve brain penetration." Has this effort taken place or been funded?
    
   Response: This proposed work is not yet funded, but we recently secured a grant to support further engineering and functionalization of PFFNB2, utilizing AAV for delivery. In meantime, to achieve improved brain penetration and expression, we have changed the AAV serotype in our ongoing studies from one used in a Nature Communications paper. We are now using the PHP.eB AAV, which has been reported to cross the blood-brain barrier (BBB) and transduce neurons more effectively in the central nervous system compared to the previously employed AAV serotype. We have injected humanized alpha synuclein mice with AAV-PHP.eB and expect to obtain results in next 4 months.
    
   

The structure of alpha-synuclein clumps (on the left) was disrupted by the nanobody PFFNB2 (as shown on the right). Credit: Xiaobo Mao